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Green tea extract: Chemistry, antioxidant propertiesand food applications A review

1756-4646/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jff.2013.08.011

* Tel.: +1 800 255 6837; fax: +1 913 764 0854.E-mail address: namal.senanayake@dupont.com.

Please cite this article in press as: Namal Senanayake, S.P.J., Green tea extract: Chemistry, antioxidant properties and food applicatioview, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.08.011

S.P.J. Namal Senanayake*

DuPont Nutrition & Health, Danisco USA Inc., Four New Century Parkway, New Century, Kansas 66031, United States

A R T I C L E I N F O A B S T R A C T

Article history:

Received 20 May 2013

Received in revised form

16 August 2013

Accepted 22 August 2013

Available online xxxx

Keywords:

Green tea extract

Antioxidant

Catechins

Flavour constituents

Lipid oxidation

Food application

Green tea is one of the most popular and extensively used dietary supplement in the United

States. Diverse health claims have made for green tea as a trendy ingredient in the growing

market for nutraceuticals and functional foods. Green tea extract contains several polyphe-

nolic components with antioxidant properties, but the predominant active components are

the flavanol monomers known as catechins, where epigallocatechin-3-gallate and epicate-

chin-3-gallate are the most effective antioxidant compounds. Additional active compo-

nents of green tea extract include the other catechins such as epicatechin and

epigallocatechin. Among these, epigallocatechin-3-gallate is the most bioactive and the

most scrutinized one. Green tea polyphenols are also responsible for distinctive aroma,

color and taste. Green tea extract can also be used in lipid-bearing foods to delay lipid oxi-

dation and to enhance the shelf-life of various food products. This review outlines the

chemistry, flavour components, antioxidant mechanism, regulatory status, food applica-

tions, and stability of green tea extract in food.

2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2. Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Flavour constituents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Antioxidant mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

6. Method of incorporation into food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

7. Application in food products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

8. Stability tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

8.1. Peroxide value (PV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

8.2. Conjugated dienes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

8.3. Thiobarbituric acid (TBA) value. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

8.4. Oil Stability Index (OSI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

8.5. Oxipres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008.6. Schaal oven storage test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

ns A re-

http://dx.doi.org/10.1016/j.jff.2013.08.011mailto:namal.senanayake@dupont.comhttp://dx.doi.org/10.1016/j.jff.2013.08.011http://dx.doi.org/10.1016/j.jff.2013.08.011http://dx.doi.org/10.1016/j.jff.2013.08.011www.sciencedirect.comhttp://www.elsevier.com/locate/jffhttp://dx.doi.org/10.1016/j.jff.2013.08.011

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8.7. Sensory evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

Tea, derived from Camellia sinensis L., is one of the most widely

consumed beverages in the world. Tea can be categorized into

three main types, depending on the level of oxidation, as

green tea, oolong tea and black tea (Chan, Soh, Tie, & Law,

2011; Velayutham, Babu, & Liu, 2008). Green tea is an ever-

green plant that grows primarily in tropical and temperate re-

gions of Asia which mainly include China, India, Sri Lanka

and Japan. It is also cultivated in several African and South-

American countries. Two primary varieties of C. sinensis are

Camellia sinensis sinensis and Camellia sinensis assamica. The

sinensis plant strain is originated from China. This strain pro-

duces green, white, black and oolong teas. On the contrary,

the assamica plant strain primarily is inhabitant to the Assam

region in Northern India. Due to enormous yields of this spe-

cific strain, it is the favored plant grown in India, Sri Lanka

and some African countries. The leaves of assamica strain

are typically used for producing black, oolong and puerh teas.

Green tea is a small shrub that can expand up to 30 feet high,

but is customarily trimmed to 25 feet when cultivated for its

leaves. The leaves are naturally murky green and glossy with

notched edges and are 25 cm broad and 415 cm long. The

flowers are white and contain bright yellow stamens. These

blossoms appear individually or as clusters. The fruits have

hard green shells with round, brown-colored seeds. These

seeds can be used to produce tea oil. Typically, flowering is

prevented during cultivation by harvesting the leaves. The

immature, light-green leaves are preferably harvested for

tea production. Mature leaves are deeper green in color than

the young leaves. Different leaf ages produce varying tea

qualities as their chemical compositions are different. Typi-

cally, the buds (tips) and the first two to three leaves are har-

vested by hand picking for processing. This process is

generally repeated every one to two weeks. Green, white,

black and oolong teas all originate from the leaves of C. sinen-

sis plant. However, the different types of tea are classified

according to the degree of fermentation that takes place dur-

ing processing: both white tea and green tea being unfer-

mented; oolong tea semi-fermented and black tea fully

fermented (Chan et al., 2011). These basic types of tea have

different quality characteristics, including appearance, aro-

ma, taste, and color. The manufacturing process of tea is

designed to either preclude, or permit tea polyphenolic com-

pounds to be oxidized by naturally occurring polyphenol

oxidase in the tea leaves. Green tea is produced by inactivat-

ing the heat-labile enzyme polyphenol oxidase in the fresh

leaves by either applying heat or steam, which prevents the

enzymatic oxidation of catechins, the most abundant

flavonoid compounds present in green tea extracts (Velayu-

tham et al., 2008). The tea leaves are then rolled, dried and

packaged.

cite this article in press as: Namal Senanayake, S.P.J., Green tea exJournal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.20

2. Chemistry

The chemical composition of tea leaves has been well docu-

mented. The main constituents of tea leaves are polyphenols

(Balentine, Wiseman, & Bouwens, 1997). The fresh tea leaves

contain caffeine (approximately 3.5% of the total dry weight),

theobromine (0.150.2%), theophylline (0.020.04%) and other

methylxanthines, lignin (6.5%), organic acids (1.5%), chloro-

phyll (0.5%) and other pigments, theanine (4%) and free amino

acids (15.5%), and numerous flavour compounds (Graham,

1992). In addition, a wide variety of other components exists,

including, flavones, phenolic acids and depsides, carbohy-

drates, alkaloids, minerals, vitamins and enzymes (Chaturve-

dula & Prakash, 2011). Tea also contains flavonols, mainly

quercetin, kaempferol, myricetin, and their glycosides. The

most favorable effects of green tea are accredited to the green

tea polyphenols, predominantly the catechins, which make

up, 2535% of the dry weight of green tea leaves (Abdel-Rah-

man et al., 2011; Balentine et al., 1997; Graham, 1992; Zaveri,

2006). The tea catechins belong to the family of flavonoids

(Yilmaz, 2006) and possess two benzene rings referred to as

the A- and B-rings. In addition, the catechin molecules con-

tain a dihydropyran heterocycle (the C-ring) that has a hydro-

xyl group on carbon 3. Moreover, the A-ring is similar to a

resorcinol moiety whereas the B-ring is similar to a catechol

moiety. The catechin molecule has two chiral centers on car-

bons 2 and 3. Hence, it has four diastereoisomers with two of

the isomers are in trans configuration, and the other two are

in cis configuration. The trans and cis isomers are referred to

as the catechin and epicatechin, respectively. These chemical

structures appear to be important for the antioxidant activi-

ties of tea polyphenols, including an ortho-3 04 0-dihydroxyl

group or 3 04 050-trihydroxyl group in the B-ring, a gallate group

located at the 3 position of the C-ring, and hydroxyl groups at

the 5 and 7 positions of the A-ring (Rice-Evans, Miller, & Pa-

ganga, 1996). Many structureactivity relationship investiga-

tions have been performed on the antioxidant activity of

flavonoids, including tea catechins (Farkas, Jakus, & Heberger,

2004; Guo et al., 1999; Harborne & Williams, 2000; Heim,

Tagliaferro, & Bobilya, 2002; Rice-Evans et al., 1996). According

to these studies, the antioxidant activity of flavonoids de-

pends substantially on the number and position of hydroxyl

groups in the molecule (Farkas et al., 2004). In addition, sev-

eral structural elements such as o-dihydroxyl catechol struc-

ture in the B-ring, the presence of unsaturation and 4-oxo

group in the C-ring are also presumed to increase the antiox-

idant activity of flavonoids. The 2,3-double bond in the C-ring

along with 4-oxo function in the C-ring facilitates electron

delocalization from the B-ring. Moreover, hydroxyl groups at

positions 3 and 5 providing hydrogen bonding to the 4-oxo

group in the C-ring is another structural feature attributed

to the antioxidant activity of flavonoids.

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Fig. 1 Chemical structures of major catechins found in

green tea extract.

J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x 3

Extracts of immature tea leaves are rich in flavanols and

their gallic acid derivatives, namely, (+)-catechin, ()-epicate-chin, (+)-gallocatechin, ()-epicatechin gallate, ()-epigalloca-techin, and ()-epigallocatechin gallate (Wanasundara &Shahidi, 2005). In addition, they contain a range of natural

green tea flavour components such as terpenes, oxygenated

terpenes, sesquiterpenes, and organic acids. As exemplified

in Fig. 1, catechins are characterized by various hydroxyl

groups on the A- and B-rings (Velayutham et al., 2008). Epicat-

echin has an ortho-dihydroxyl group in the B-ring at carbons

3 0 and 4 0 and a hydroxyl group at carbon 3 on the C-ring

(Fig. 1). Epigallocatechin differs from epicatechin in that it

has a trihydroxyl group at carbons 3 0, 4 0, and 5 0 on the B-ring

(Yilmaz, 2006). Epicatechin gallate differs from epicatechin in

its gallate moiety positioned at carbon 3 of the C-ring (Yilmaz,

2006). However, epigallocatechin gallate is composed of tri-

hydroxyl groups on the B-ring and a gallate moiety located

at carbon 3 of the C-ring (Yilmaz, 2006). In epigallocatechin

gallate, these trihydroxyl groups are located at carbons 3 0, 4 0

and 50 on the B-ring. Yilmaz (2006) has reviewed the chemis-

try and application perspectives of green tea, especially in

relation to using their catechin components. The relative con-

tent of green tea catechins depends on how the leaves are

processed prior to drying. Fermentation and heating of tea

leaves during the manufacturing process may cause polymer-

ization of monopolyphenolic catechins, leading to conforma-

tional changes and thus modifying their properties (Cabrera,

Artacho, & Gimenez, 2006).

Green tea contains significantly higher amounts of poly-

phenols as compared to black or oolong tea, owing to differ-

ences in the processing of tea leaves following harvest. For

green tea, freshly harvested tea leaves are steamed at high

temperatures and dried to deactivate the polyphenol oxidases

and, as a result, preventing the oxidation of catechins and

maintaining the polyphenols in their monomeric forms. In

addition to preventing polyphenol oxidation, the steaming

process also protects against enzymatic degradation of vita-

mins. Through separation, purification, concentration and

the drying processes, highly concentrated green tea catechin

extract with low flavour intensity can be produced. Black tea,

Please cite this article in press as: Namal Senanayake, S.P.J., Green tea exview, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.20

on the other hand, is produced by extended fermentation of

tea leaves, which results in, the development of polymeric

compounds including thearubigins and theaflavins (Zaveri,

2006). Black tea contains predominantly gallates of epicate-

chin. Oolong tea, a partially fermented product, contains a

mixture of the monomeric polyphenols and high-molecular-

weight theaflavins (Graham, 1992; Zaveri, 2006). All three vari-

eties of tea contain significant quantities of caffeine (36%)

which is unaffected by different processing conditions (Chu,

1997; Zaveri, 2006). Furthermore, all three varieties of tea con-

tain ()-epicatechin, ()-epigallocatechin, ()-epicatechin gal-late and ()-epigallocatechin gallate, with the exception ofcatechin (Khokhar, Venema, Hollman, Dekker, & Jonge,

1997). Epicatechin gallate and epigallocatechin gallate are

considered to be the main catechins found in black tea (Oban-

da, Owuor, & Mangoka, 2001; Yilmaz, 2006). Epigallocatechin

gallate is the most copious catechin component present in

the leaves of green, black and oolong teas (Graham, 1992). In

general, the contents of epigallocatechin gallate in green

and oolong teas range from 22 to 53 mg/g of commercial teas

(Zuo, Chen, & Deng, 2002). However, epigallocatechin gallate

content in black teas is about 4.0 mg/g of commercial teas

(Zuo et al., 2002).

The stability of green tea catechins depends on the pH and

temperature. In acidic solutions (pH < 4), green tea catechins

exhibit exceptional stability; in alkaline solutions (pH > 8),

however, they are extremely unstable (Zhu, Zhang, Tsang,

Huang, & Chen, 1997). The study conducted by Zhu et al.

(1997) demonstrated that ()-epigallocatechin gallate and()-epigallocatechin were more unstable than ()-epicatechinand ()-epicatechin gallate in a basic solution, giving a plau-sible explanation to the fact that partial absorption of green

tea catechins in red blood cells of mice is attributed to the

instability of ()-epigallocatechin gallate and ()-epigallocate-chin in the intestine where the pH is neutral or alkaline. In a

high temperature environment, green tea catechins are not

very stable. Heating may cause the conversion of green tea

catechins to their corresponding isomers, a process known

as epimerization. For example, as epimerization can occur

at high temperature, epigallocatechin gallate in green tea ex-

tract may convert to its epimer component gallocatechin gal-

late. Heat treatments decrease the antioxidant activity of

green tea catechins due to oxidation, thermal degradation,

epimerization and polymerization (Ananingsih, Sharma, &

Zhou, 2013).

3. Flavour constituents

The taste and flavour of tea is governed by key chemical com-

ponents which are polyphenols, caffeine, organic acids and

volatile terpenes (Borse, Rao, Nagalakshmi, & Krishnamurthy,

2002). The characteristic taste of green tea extract is made up

of a mixture of bitterness, astringency, meaty (umami) taste,

sweetness and slight sourness. The compounds, which con-

tribute to, characteristic tea taste, are polyphenolic com-

pounds, amino acids and caffeine (Yamanishi, 1995). Volatile

compounds such as terpenoids, alcohols and carbonyl com-

pounds contribute to the aroma of tea. Volatile fractions of var-

ious green teas reported having numerous active aroma

compounds which are responsible for nutty, floral, fruity,

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4 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x

meaty, popcorn-like, metallic, potato, green, cucumber-like,

and hay-like characteristics (Kumazawa & Masuda, 2002). In

a study conducted by Pripdeevech and Machan (2011), the vol-

atile flavour components of different teas growing in Thailand

were evaluated. These authors reported that the utmost vola-

tile flavour components in green tea were found to be hotrie-

nol, geraniol and linalool. In addition, green Assam tea was

reported to contain linalool, geraniol and a-terpineol as the

key flavour constituents (Pripdeevech & Machan, 2011).

In a classic study to understand the taste characteristics of

different types of catechins, Nakagawa (1970) found that (+)-

catechin, ()-epicatechin, and ()-epigallocatechin were bit-ter with a sweet aftertaste while ()-epicatechin gallate and()-epigallocatechin gallate were bitter and astringent. In afollow up study to determine the chemical components that

affect the taste attributes of green tea in Japan, Nakagawa

(1975) discovered that teas containing higher content of cate-

chins and amino acids scored much higher in taste tests by

consumers. His results indicated that the astringency and bit-

terness of green tea was primarily determined by the content

of catechins and other phenolic compounds. The taste of oo-

long tea appears to be unique. According to Chaturvedula and

Prakash (2011), the mellowness and sweetness of oolong tea

are attributed to non-oxidized catechins, thearubigins, some

secondary polyphenolic compounds, caffeine, free amino

acids and related sugars. The astringency of oolong tea is low-

er, and the sweet taste is stronger than those of green tea. The

distinctive taste of black tea is due to the combination of sev-

eral attributes such as bitterness, astringency, sweetness,

malty taste, green/grassy taste, caramel-like and hay-like

characteristics (Alasalvar et al., 2012). Yang and Landau

(2000) reported that the theaflavins are crucial to the charac-

teristic color and taste of black tea. The theaflavins contain

numerous compounds including, theaflavin, theaflavin-3-gal-

late, theaflavin-3 0-gallate and theaflavin-3,3 0-digallate (Chan

et al., 2011). The key astringent taste compounds in black

tea are catechins, theaflavins, and flavonol glycosides (Schar-

bert, Holzmann, & Hofmann, 2004). Although compounds in

all three categories are detected as astringent, flavonol glyco-

sides are the most abundant contributors of astringency in

black tea (Scharbert et al., 2004).

Amino acids are the components in green tea that contrib-

ute full-bodied umami flavour and sweetness. Of these amino

acids, more than 50% are theanine, which is unique to tea

(Chaturvedula & Prakash, 2011). The amino acid theanine

has a chemical structure somewhat similar to that of gluta-

mine, with its distinct attribute being a refined, rich flavour

and sweetness. Amino acids other than theanine present in

tea leaves are glutamine, alanine, asparagine, arginine and

serine. The umami taste of green tea is shown to be due to

theanine and serine. It is believed that 70% of the umami

taste in Japanese green tea is due to theanine (Kaneko,

Kumazawa, Masuda, Henze, & Hofmann, 2006). By conducting

molecular and sensory studies, Kaneko et al. (2006) concluded

that theanine, gallic acid, theogallin and succinic acid are the

main compounds responsible for umami taste of green tea.

Besides catechins and caffeine, some amino acids, in particu-

lar, arginine and alanine also contribute to the bitterness of

green tea (Chaturvedula & Prakash, 2011).

Please cite this article in press as: Namal Senanayake, S.P.J., Green tea exview, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.20

4. Antioxidant mechanism

Lipid oxidation and development of rancidity is a major chal-

lenge for food manufacturers, reducing shelf-life and altering

the quality and nutritional value of their products. Autoxida-

tion, the most common process leading to oxidative deterio-

ration of food lipids, is a free radical chain reaction that

proceeds through three distinct stages of initiation, propaga-

tion and termination. The generation of primary free radicals

is facilitated by the presence of oxidation initiators such as

light, heat, ionizing radiation, transition metals, metallopro-

teins, oxidants, various homolysis-prone substances and en-

zymes. Lipid hydroperoxides have been identified as

primary products of autoxidation. Typically, lipid hydroperox-

ides are tasteless and odorless. The decomposition of hydro-

peroxides yields aldehydes, ketones, alcohols, hydrocarbons

and acids, which are known as secondary oxidation products

of lipids. In many cases, these compounds are responsible for

off-flavours and off-odors in food. Antioxidants are of interest

to the food industry because they can delay development of

oxidative rancidity in food. An antioxidant is a substance that

retards lipid oxidation either by inhibiting initial free radical

formation or by preventing them from producing more free

radicals which can disseminate the oxidation reaction. These

substances help preserve foods by delaying development of

rancidity, deterioration and discoloration due to lipid oxida-

tion. There are two main categories of antioxidants in relation

to their mechanism of action: primary antioxidants and sec-

ondary or preventive antioxidants. Primary antioxidants dis-

rupt the oxidative free radical chain reaction by donating

electrons or hydrogen atoms from the phenolic hydroxyl

groups and, therefore, stabilize lipid free radicals, as a result,

inhibit or slow down the initiation phase and disrupt the

propagation stage of autoxidation. Secondary antioxidants

deactivate singlet oxygen, chelate metal ions (i.e., iron, cop-

per), absorb ultraviolet radiation, scavenge oxygen and help

regenerate primary antioxidants. For better effectiveness, pri-

mary antioxidants are often used in combination with sec-

ondary antioxidants.

Tea polyphenols, mainly flavonoids, are well-known for

their antioxidant properties. The antioxidant activity of green

tea polyphenols is primarily attributed to the combination of

aromatic rings and hydroxyl groups that assemble their

chemical structure and consequently binding and neutraliza-

tion of lipid free radicals by these hydroxyl groups. Numerous

studies have demonstrated that polyphenols and tea cate-

chins are exceptional electron donors and effective scaveng-

ers of physiologically relevant reactive oxygen species

in vitro, including superoxide anions (Guo et al., 1999; Micha-

lak, 2006; Nakagawa & Yokozawa, 2002; Nanjo et al., 1993;

Velayutham et al., 2008), peroxyl radicals, and singlet oxygen

(Guo et al., 1999; Michalak, 2006). Catechins also exhibit anti-

oxidant activity via chelating redox active transition-metal

ions. Polyphenolic compounds, possess hydroxyl and car-

boxyl groups, are able to bind particularly iron and copper

(Michalak, 2006). There is another mechanism underlying

the antioxidant ability of plant polyphenols, including tea cat-

echins. Transition metal ions typically have the ability to ini-

tiate free radical chain oxidations by decomposing lipid

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J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x 5

hydroperoxides (LOOH) by the hemolytic cleavage of the OO

bond and producing lipid alkoxyl radicals. Phenolic antioxi-

dants, including tea catechins, inhibit lipid peroxidation by

binding these lipid alkoxyl radicals. This activity depends on

the structure of the molecules, and the number and position

of the hydroxyl group in the molecules (Milic, Djilas, & Cana-

danovic-Brunet, 1998). Green tea catechins also exhibit anti-

oxidant activity through inhibiting pro-oxidant enzymes

and inducing antioxidant enzymes (Velayutham et al., 2008).

The antioxidant activity of these catechins and the deriv-

atives showed a marked difference depending on the sub-

strate used for evaluation (Wanasundara & Shahidi, 2005).

Green tea catechins, which performed like other hydrophilic

antioxidants such as Trolox (a water-soluble analog of a-

tocopherol) and ascorbic acid, have been shown to be active

antioxidants in bulk oils, but were prooxidants in the corre-

sponding oil-in-water emulsions (Frankel, Huang, & Aesc-

bach, 1997; Frankel, Huang, Kanner, & German, 1994). In

corn oil triacylglycerols system that was oxidized at 50 Cthe antioxidant activities of epigallocatechin, epigallocate-

chin gallate and epicatechin gallate were superior to those

of epicatechin or catechin (Huang & Frankel, 1997). These cat-

echins have also been highly successful in delaying oxidation

of polyunsaturated fatty acids-rich marine and vegetable oils

(Wanasundara & Shahidi, 1998). In the lecithin-containing lip-

osomes system, epigallocatechin gallate was superior to other

compounds such as epicatechin, epigallocatechin, epicate-

chin gallate, and catechin (Huang & Frankel, 1997). In the

oil-in-water emulsions, however, all catechin compounds

demonstrated pro-oxidant activity (Huang & Frankel, 1997).

The improved antioxidant activity observed for tea catechins

in liposomes compared to emulsions has been explained by

the greater affinity of the polar catechins toward the polar

surface of the lecithin bilayers, thus affording better protec-

tion (Huang & Frankel, 1997). In a study conducted by Zhong

and Shahidi (2011), epigallocatechin gallate was structurally

modified to improve its lipophilicity via esterification of epi-

gallocatechin gallate with selected fatty acids, such as stearic

acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid

(DHA). The lipophilized derivatives so produced exhibited

greater antioxidant activity than the original epigallocatechin

gallate molecule.

Several investigators have speculated the mechanism of

antioxidant action of (+)-catechin using some oxidation mod-

el studies (Hirose, Yamamoto, & Nakayama, 1990; Koketsu,

1997; Zhu et al., 2000). As indicated by the proposed mecha-

nism, (+)-catechin molecule can scavenge four lipid free rad-

icals per molecule (Hirose et al., 1990; Koketsu, 1997). The

antioxidant activity of individual tea polyphenols in different

model systems showed a proportional relationship to the

number of hydrogen radical donors of catechins. A synergistic

effect has been observed between green tea catechins and

ascorbic acid and a-tocopherols (Murakami, Yamaguchi,

Takamura, & Matoba, 2003). Catechin, a monomeric flavanol,

is reported to have hydroxyl, peroxyl, superoxide and 1,1-di-

phenyl-2-picrylhydrazyl (DPPH) radical scavenging activities

(Bors & Michel, 1999; Fukumoto & Mazza, 2000). Moreover,

tea catechins have the ability to chelate iron in food model

systems (Tang, Kerry, Sheehan, & Buckley, 2002). Nakao, Taki-

o, and Ono (1998) reported that the peroxyl radical scavenging

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activities of catechin, epicatechin and epicatechin gallate

were ten times higher than those of L-ascorbate and beta-car-

otene. In another study, Nanjo et al. (1996) documented that

DPPH radical scavenging activities of catechin and epicate-

chin are less than epigallocatechin, epicatechin gallate, and

epigallocatechin gallate. As expected, epicatechin is another

monomeric flavanol (Yilmaz, 2006) came across in green tea.

Reports have shown that epicatechin is capable of scavenging

hydroxyl radicals, peroxyl radicals, superoxide radicals, and

DPPH radicals (Bors & Michel, 1999; Fukumoto & Mazza,

2000; Liu, Ma, Zhou, Yang, & Liu, 2000; Yilmaz, 2006). Yashin,

Yashin, and Nemzer (2011) examined the antioxidant activity

of different types of teas that include green, oolong, black and

pu-erh teas. The antioxidant activity of teas descends in the

following order: green > oolong > black > puerh tea. Chan

et al. (2011) evaluated the role of non-polymeric phenolic

(NP) and polymeric tannin (PT) constituents in the antioxi-

dant and antibacterial properties of six brands of green, black,

and herbal teas. Extraction yields of the products ranged from

12% to 23%. Much higher fractionation yields more NP con-

stituents (7081%) than PT constituents (111%), which sug-

gested that, the former are the principal tea components. In

general, the antioxidant properties of green tea extracts were

stronger than those of black and herbal teas (Chan et al.,

2011). For all six brands tested, antioxidant properties of PT

fractions were significantly higher than crude extracts and

NP fractions. Although PT constituents have stronger antiox-

idant and antibacterial properties, they constitute only a min-

or component of the teas (Chan et al., 2011).

Green tea polyphenols have been shown to be effective

against beta-carotene oxidation. For example, tea catechins

have been able to demonstrate an anti-discoloring effect on

beverages and margarine containing beta-carotene (Koketsu,

1997; Unten, Koketsu, & Kim, 1997). It was suggested that

the discoloration of beta-carotene and the oxidation of unsat-

urated fatty acids progressed by the same mechanism. In par-

ticular, carboncarbon double bonds in beta-carotene and

unsaturated fatty acids are attacked by radicals, becoming

hydroperoxides. Green tea catechins have the ability to sup-

press the degradation of double bonds as radical scavengers.

It was also suggested that the hydroxyl group at the 5 0-posi-

tion of the B-ring of the catechin structure mostly contributed

to the anti-discoloring effect. Hence, as described above, it

was speculated that green tea polyphenols delayed the degra-

dation of beta-carotene by acting as antioxidants following

the same mechanism. Among the individual green tea poly-

phenols examined, gallocatechin gallate, epigallocatechin

gallate, epigallocatechin, and gallocatechin showed strong

anti-discoloring effect while epicatechin and catechin

showed almost no activity, and gallic acid had moderate

activity (Unten et al., 1997).

5. Regulatory status

In the United States, green tea extract may be used as flavour

agent with antioxidative properties in various fats, oils and

foods containing fats and oils. Green tea has a history of safe

use as food flavouring and is known for its antioxidative func-

tionality as a secondary effect. Green tea extract is well

known in Asian food. According to the United States Code

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6 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x

of Federal Regulations (21 CFR 101.22, and 21 CFR 182.10), the

term natural flavour or natural flavouring means the essen-

tial oil, oleoresin, essence or extractive, protein hydrolysate,

distillate, or any product of roasting, heating or enzymolysis,

which contains the flavouring constituents derived from a

spice, fruit or fruit juice, vegetable or vegetable juice, edible

yeast, herb, bark, bud, root, leaf or similar plant material,

meat, seafood, poultry, eggs, dairy products or fermentation

products thereof, whose significant function in food is fla-

vouring rather than nutritional (U.S. Food and Drug Adminis-

tration, 2012). Natural flavours include the natural essence or

extractives obtained from plants listed in sections CFR 182.10,

182.20, 182.40, and 182.50 and part 184 of this chapter, and the

substances listed in section 172.510 of this chapter (U.S. Food

and Drug Administration, 2012). Thus, in the United States,

green tea extract may be labeled as natural flavouring. In

European Union (EU), the definition of natural flavouring sub-

stances, as stated in Council Directive 88/388/EEC, applies to

green tea extracts when used as flavourings, and they can

be used in all food applications, unless regulations restrict

the use of flavourings in specific foods (Official Journal of

the European Union, 2008). Certain green tea extracts are also

regarded as traditional foods in the EU, however, market

clearance of green tea extracts depends on the intended use

as well as the product profile. If the primary function high-

lighted is the antioxidant properties, the green tea extract is

then regulated as an antioxidant and must be specifically ap-

proved. Results of various investigations have confirmed that

green tea polyphenolic compounds are largely responsible for

distinctive aroma, color and taste (Borse et al., 2002; Chat-

urvedula & Prakash, 2011; Pripdeevech & Machan, 2011;

Yamanishi, 1995). Hence, various compounds of green tea ex-

tract that contribute to the antioxidant effects also contribute

to flavour properties. The combination of these functional-

ities may allow innovative solutions such as designing spe-

cific green tea extract products to target certain food

applications. As green tea extract contributes to the flavour

profile of food matrix and helps retard lipid oxidation, it can

be successfully used in a wide array of oils, fats, and foods

containing fats and oils.

6. Method of incorporation into food

Proper incorporation of green tea extract into food products is

imperative to ensure that antioxidant components are thor-

oughly dissolved and/or homogeneously dispersed in the

food matrix. As only a small amount of green tea extract

may be required for adequate shelf-life extension in food,

the method of application may determine the achievement

of the antioxidant effectiveness. The selection of application

methods may depend on the type of food products, process-

ing methods, and the availability of the equipment used for

food processing. Commercial extracts of green tea are mainly

available in a fine-grained powder form. Green tea extract can

be solubilized in water prior to addition into foods. Water-sol-

uble green tea extract has a low viscosity for easy spraying

and homogeneous distribution. Moreover, remarkably little

energy is required to produce the dispersion in water. On

the other hand, green tea extract in powder form is often pre-

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ferred in certain formulations containing dry ingredients as

this enables easier blending. The powder carrier aids the

incorporation and homogeneous distribution of the green

tea extract. Where dry ingredients are part of the formulation,

a powdered green tea extract is often preferred due to easier

blending. Conversely, Green tea extract can be dissolved in a

food-grade solvent to produce an oil soluble or dispersible li-

quid product. Liquid forms of green tea extracts may be added

to food products by direct addition into oils and fats. The fat

or oil may be heated to an elevated temperature (i.e., 40

60 C) while stirring. Liquid green tea extract should then beadded slowly into melted fat or oil. After addition is com-

pleted, agitation should be continued for an additional period

of time for complete and uniform distribution of green tea ex-

tract in fats and oils.

7. Application in food products

There is growing consumer interest in the green tea extract-

supplemented products in recent years. Green tea extract

has been used in a variety of food products including bread

(Wang & Zhou, 2004), biscuits, dehydrated apple (Lavelli, Van-

taggi, Corey, & Kerr, 2010) and various meat products (Mit-

sumoto, OGrady, Kerry, & Buckley, 2005; OSullivan, Lynch,

Lynch, Buckley, & Kerry, 2004; Tang, Kerry, Sheehan, & Buck-

ley, 2001). The main application areas of green tea extract

are summarized in Table 1. In internal tests with fat spreads,

green tea extract showed comparable antioxidant perfor-

mance to conventional synthetic antioxidant tert-butylhydro-

quinone (TBHQ) and may be more cost-effective than other

natural sources of antioxidants. This makes it ideal for both

full-fat margarines as well as oxidation-sensitive products

that are low in fat, free of trans fatty acids or have a high con-

tent of polyunsaturated fatty acids. In this study, samples of

40% low-fat spread were produced with green tea extract con-

taining 60 ppm total catechins or 100 ppm TBHQ and stored at

5 C for 13 weeks. A control sample contained no antioxidantsolution. In an accelerated shelf-life test, a comparison of

green tea extract and TBHQ revealed similar performance.

Lipid oxidation is one of the primary causes of quality

deterioration in meat and poultry products. Red meats and

poultry are typically highly susceptible to lipid oxidation

due to their high fat content (Yilmaz, 2006) and a high propor-

tion of polyunsaturated fatty acids. These highly unsaturated

phospholipids present in meat and poultry products are

responsible for the development of off-flavours and off-odors

after cooking and subsequent refrigerated storage. This off-

flavour development, also referred to as warmed-over-flavour

(WOF), frequently causes consumers to reject freshly cooked

meat and poultry products. Therefore, these products should

be protected by natural sources of antioxidants such as green

tea extract to protect against WOF.

In a study conducted by DuPont Nutrition & Health labora-

tories, both control and green tea extract-stabilized turkey

burgers were made using standard industry practice. Green

tea extract was added, at a dosage level of 50100 ppm. Sam-

ples were evaluated for thiobarbituric acid reactive sub-

stances (TBARS) formation, reported as malondialdehyde (a

secondary product of lipid oxidation) equivalents. Results

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Table 1 Prospective applications of green tea extract.

Application Specific oxidation issues

Raw meats Oxidation of red meat pigments, resulting in

an undesirable brown color

Cooked meat, poultry and

seafood products

Susceptible to oxidation, resulting in a

warmed-over flavor, discoloration and protein degeneration

Ready-to-eat meals Reheating of meat promotes the oxidation process

Cereals, bakery products, confectioneries,

snack foods, nuts and nut products

Products are susceptible to oxidation due to long shelf-life requirements

Oil-in-water emulsions

(mayonnaise, salad dressings, soups and sauces)

Large oilwater interface and complex food matrix increase susceptibility

to lipid oxidation

Water-in-oil emulsions

(margarine and fat spreads)

Large wateroil interface and complex food matrix increase susceptibility

to lipid oxidation

Vegetable oils, marine oils,

frying oils, and shortenings

Low oxidative stability, due to trans fatty acid regulations, increases the need for

enhanced antioxidant protection

Beverages (carbonated and non-carbonated beverages,

energy drinks, soft drinks, and juices)

Products are susceptible to oxidation due to long shelf-life

requirements

Fig. 2 TBARS of roasted turkey burgers during storage at

4 C.

Fig. 3 Effect of green tea extract and BHA on the inhibition

of TBARS of roasted turkey burgers during storage at 4 C.

Fig. 4 Hexanal contents of roasted beef burgers during

storage at 4 C.

J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x 7

showed that oxidation in roasted turkey burgers was signifi-

cantly retarded using green tea extract, at levels of 50 to

100 ppm. Samples supplemented with green tea extract had

lower levels of TBARS during storage as compared to control

with nothing added (Fig. 2). In another study conducted by

DuPont Nutrition & Health, the efficacy of green tea extract

was compared with equivalent dosages of butylated hydroxy-

anisole (BHA) and revealed that a dosage of 200 ppm of green

tea extract simply provided the same antioxidant protection

as 40 ppm BHA in chilled (4 C) roasted turkey burgers duringstorage (Fig. 3). The sensory evaluation of the products further

confirmed the advantage of using green tea extract compared

to BHA. In a different study conducted by DuPont Nutrition &

Health, the addition of 150 ppm green tea extract was ade-

quate to inhibit hexanal development (a secondary product

of lipid oxidation) during 13 days of storage in roasted beef

burgers (Fig. 4). This study compared the antioxidant capacity

obtained from 150 ppm green tea extract with that obtained

from 375 ppm rosemary extract. Green tea and rosemary

extract-supplemented samples had low levels of hexanal

throughout storage when compared with the control contain-

ing nothing added. Furthermore, green tea extract-supple-

mented sample had lower levels of hexanal as compared to

the rosemary extract-treated sample throughout storage

(Fig. 4).

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The inhibitory effect of green tea extract on the oxidation

of meat lipids was also shown by other investigators. Studies

conducted by Tang et al. (2001) and Mitsumoto, OGrady,

Kerry, and Buckley (2005) showed a high antioxidant activity

of green tea catechins in beef and chicken meat. In another

study, OSullivan, Lynch, Lynch, Buckley, and Kerry (2004)

evaluated the effect of several natural extracts on oxidative

stability of chicken nuggets during refrigerated (4 C) storage.Nuggets were supplemented with rosemary extract (at

tract: Chemistry, antioxidant properties and food applications A re-13.08.011

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Fig. 5 Color of raw ground beef patties treated with green tea extract and rosemary extract compared to an unprotected

control sample.

8 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x

1000 ppm), sage extract (at 1000 ppm), or tea catechins (at

100 ppm). Their results indicated that the addition of natural

extracts, including tea catechins, reduced lipid oxidation in

chicken nuggets both in the presence and absence of salt.

However, when sodium tripolyphosphate (STPP) was incorpo-

rated into the same product system, the efficacy of natural ex-

tracts was significantly reduced.

In addition to providing antioxidant activity and natural

flavor attributes, green tea extract can also help improve the

color stability of fresh meat products. Fresh meat color de-

pends on myoglobin that stores oxygen for aerobic metabo-

lism in the muscle. Iron is a pivotal player in meat color.

One of the defining factors of meat color is the chemical state

of iron. Oxymyoglobin gives meats the red color. The brown-

ish color is due to metmyoglobin formed by oxidation of oxy-

myoglobin. However, how green tea extract influences the

color cycle in meat products is not well understood. Studies

conducted by DuPont Nutrition & Health have shown that

the red color of raw ground beef patties (20% fat) was signifi-

cantly improved by the addition of rosemary extract

(1000 ppm) and green tea extract (250 ppm) compared to the

development of browning in control samples (Fig. 5). All sam-

ples were stored at 02 C for 22 days and packaged undermodified atmosphere (80% oxygen and 20% carbon dioxide)

packaging conditions. Determination of redness of the meat

(a value using Minolta Colorimeter) corresponded with the

development as the visible red color. The meat had lost

almost if not all visible red color when the a value reached

approximately 12. The control reached this point around

day 9, and the treated samples around day 16. This shows

that 1000 ppm rosemary extract and 250 ppm green tea

extract exhibited good protection of color for several days be-

yond the control sample, and there was a minimal difference

between the two treatments (Fig. 5). Sensory evaluation of the

burgers showed that green tea extract had no unfavorable fla-

vour impact on the finished burgers where slight rosemary

flavour could be detected in the samples containing rosemary

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extract. In addition, green tea extract contributed to meaty

(umami) taste in finished burgers (data not shown). In flavour

sensitive meat systems, green tea extract would be a more

viable option than the rosemary extract due to similar effec-

tiveness, but lower flavour impact. A study by Moawad, Abo-

zeid, and Nadir (2012) has shown that the use of tea catechins

in combination with nitrite was more effective in keeping

quality of dry-cured sausages during refrigerated storage as

indicated by attractive red color (a* value) as compared to

samples treated with nitrite. In another study conducted by

DuPont Nutrition & Health laboratories, cured ham slices con-

taining 500 ppm sodium ascorbate and 300 ppm green tea ex-

tract enhanced color stability (from 14 days to 25 days) by

maintaining the redness remarkably efficiently as compared

to a standard ham with 500 ppm sodium ascorbate. The

ham slices were packaged under MAP conditions (80% oxygen

and 20% carbon dioxide) and stored at 5 C for a period of38 days. This was shown visually and measured by a Videom-

eterLab 2 High Performance Multispectral Imaging by

recording a-values. The a-value of 17 was defined as the work-

ing limit for redness acceptability of cured ham.

Results of numerous investigations have also confirmed the

antioxidant properties of green tea catechins in various lipid

and food model systems (Amarowicz & Shahidi, 1995; Chen,

Chan, Ma, Fung, & Wang, 1996; Frankel et al., 1997; Huang &

Frankel, 1997; Koketsu & Satoh, 1997; Matsuzaki & Hara,

1985; Mildner-Szkudlarz, Zawirska-Wojtasiak, Obuchowski, &

Goslinski, 2009; Roedig-Penman & Gordon, 1997; Wanasundara

& Shahidi, 1998). When green tea catechins were added to noo-

dles and to the edible oils (soybean, fish, lard) they were able to

improve the oxidative stability of the fried product and the oil

used for frying (Frankel et al., 1997; Koketsu & Satoh, 1997). In a

study with marine oils such as seal blubber oil and menhaden

oil, both antioxidant and pro-oxidant effects of green tea ex-

tracts have been reported (Wanasundara & Shahidi, 1998).

Green tea extracts demonstrated a pro-oxidant effect in both

oils evaluated, possibly due to the presence of their chlorophyll

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J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x 9

components. Consequently, in follow-up studies, a column

chromatography was utilized to eliminate chlorophyll com-

pounds from green tea extracts. The dechlorophyllized green

tea extract so produced was incorporated into both seal blub-

ber and menhaden oils at various levels. The antioxidant activ-

ity of dechlorophyllized green tea extract was compared with

that of the butylated hydroxyanisole (BHA), butylated

hydroxytoluene (BHT), TBHQ and a-tocopherol. At the levels

of P200 ppm, dechlorophyllized green tea extract showedexceptional antioxidant activity in both oils examined. More-

over, the efficacy of dechlorophyllized green tea extract was

superior to that of the BHA (at 200 ppm), BHT (at 200 ppm)

and a-tocopherol (at 500 ppm), but inferior to that of TBHQ

(at 200 ppm). In several other studies, antioxidant activity of

tea polyphenols in vegetable oils and animal fats was exam-

ined (Chen et al., 1996; Koketsu, 1997; Koketsu & Satoh,

1997). Tea polyphenols evidently demonstrated an inhibitory

effect on oxidation of lard, and this effect was dose-depen-

dent. Furthermore, there was a distinct suppressive effect of

tea polyphenols on oxidative rancidity of soybean oil (Koketsu,

1997). When green tea extract was compared with BHA and

tocopherols, green tea catechins showed better antioxidant

activity in fish oil (Koketsu & Satoh, 1997). The ethanol extracts

of green tea catechins strongly inhibited the oxidation of cano-

la oil at 100 C as shown by measurement of oxygen consump-tion, whereas extracts of black tea showed little to no

antioxidant activity (Chen et al., 1996). Crude green tea extract

powders were more effective than a-tocopherol and BHA in

lard under conditions of the active oxygen method (AOM) at

97.8 C (Matsuzaki & Hara, 1985). In a b-carotene-linoleatemodel system, ()-epicatechin-3-gallate exhibited the stron-gest antioxidative activity whereas ()-epigallocatechin hadthe weakest performance (Amarowicz & Shahidi, 1995). Fur-

thermore, the antioxidative efficacy of ()-epicatechin and()-epigallocatechin-3-gallate was similar and in betweenthose of ()-epicatechin-3-gallate and ()-epigallocatechin.In another study, Mildner-Szkudlarz et al. (2009) examined

the feasibility of using green tea extract to improve the oxida-

tive stability of biscuits during storage. The antioxidant activ-

ity of green tea extract was compared with commonly used

synthetic antioxidant BHA. Phenolic compounds of green tea

extract had significantly higher DPPH radical scavenging activ-

ity than BHA. However, both green tea extract and BHA were

effective inhibitors of the decomposition of hydroperoxides.

Moreover, biscuits treated with green tea extract had better

sensory profile during storage as compared to samples con-

taining BHA.

8. Stability tests

Oxidative stability is a decisive factor in the processing and

marketing of oils, fats, and lipid-bearing foods. Methods for

determining oxidative stability are, therefore, essential partic-

ularly when green tea extract is being evaluated for its effec-

tiveness in delaying lipid oxidation in these products.

Consequently, many protocols have been developed in at-

tempts to assess the effectiveness of green tea extract in food.

Oxidative stability of green tea extract-supplemented food

products may be determined by storing samples at normal

use conditions and examining them periodically for organo-

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leptic (odor and flavour) changes or by testing them chemically

for oxidative rancidity. Although normal use conditions are

ideal, this protocol may be too slow to be of practical value.

Current methods for determining the effectiveness of green

tea extract against lipid oxidation in food are discussed in this

section.

8.1. Peroxide value (PV)

The peroxide value (PV), an indicator of primary products of

lipid oxidation, has been the most commonly employed

chemical assay for evaluating oxidative stability of fats and

oils. In this test, the melted fat or oil sample is titrated with

a mixture of glacial acetic acidchloroform (3:2, v/v) and sat-

urated potassium iodide (KI) solution. As hydroperoxides

present in the sample oxidize iodide to iodine, the liberated

iodine is then titrated with standardized solution of sodium

thiosulfate to an end-point. This method is highly empirical,

and any discrepancy in the test procedure may result in a var-

iation of results. The PV is calculated by determining all sub-

stances which oxidize KI, in terms of milli-equivalents of

peroxides per kilogram of sample (Shahidi & Zhong, 2005).

These substances are generally assumed to be peroxides or

other similar products of oil oxidation. The content of hydro-

peroxides can be calculated directly by titration of iodine so

produced. In order to avoid the use of chloroform, the Amer-

ican Oil Chemists Society (AOCS, 2003) has developed an

alternative method, which uses isooctane as solvent (AOCS,

2003; Official Method Cd 8b-90), although the method is lim-

ited to PV of less than 70 meq/kg as described in the AOCS

guidelines. The results of this test are linked to flavor precur-

sors such as hydroperoxides, but not to the actual flavour

compounds. In an internal study, samples of 40% low-fat

spreads were produced with green tea extract containing

60 ppm total catechins or 100 ppm TBHQ and stored at 5 Cfor 13 weeks. A control sample contained no antioxidant solu-

tion. While the hydroperoxides content has significantly in-

creased in the control sample, the samples containing green

tea extract or TBHQ had significantly fewer hydroperoxides

after 15 weeks of storage. In another study, Mustafa (2013)

demonstrated that green tea extract inhibited lipid hydroper-

oxides formation in ground beef during refrigerated storage

(Mustafa, 2013).

8.2. Conjugated dienes

Oxidation of highly unsaturated fatty acids is associated with

an increase in the ultra violet (UV) absorption of the product.

During lipid oxidation, double bonds in unsaturated fatty

acids are changed from non-conjugated to conjugated double

bonds. This process takes place almost immediately after

hydroperoxides have been formed (Gunstone & Norris,

1983). These conjugated diene hydroperoxides have a UV

maximum at 230240 nm, with a strong characteristic absorp-

tion at 232 nm. Formation of conjugated dienes is an indicator

of detection of primary changes in lipid oxidation. Compared

to peroxide value determination, the measurement of conju-

gated dienes is much simpler and faster and does not rely on

chemical reactions (Shahidi & Zhong, 2005) or formation of

color development. The inhibition of formation of conjugated

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10 J O U R N A L O F F U N C T I O N A L F O O D S x x x ( 2 0 1 3 ) x x x x x x

dienes by green tea extract or other sources of antioxidants

can be used as means of assessing antioxidant performance

in food. Formation of conjugated dienes is reported to corre-

late well with peroxide values during the early stages of lipid

oxidation (Wanasundara, Shahidi, & Jablonski, 1995). During

Internal testing on 40% fat spreads, the addition of 300 ppm

green tea extract was adequate to inhibit conjugated dienes

formation during 13 weeks of storage at 5 C. In another inter-nal study, samples of 40% low-fat spreads were prepared with

green tea extracts containing 40 ppm catechins, and 60 ppm

catechins or 100 ppm TBHQ solution and stored at 5 C for15 weeks. The addition of green tea extract had a dose-depen-

dent effect on the inhibition of conjugated dienes. Moreover,

the addition of green tea extract with 60 ppm catechins and

100 ppm TBHQ inhibited the development of conjugated

dienes most effectively throughout the storage period.

8.3. Thiobarbituric acid (TBA) value

TBA is one of the most widely used methods of assessing the

extent of lipid oxidation in foods. The TBA value is typically

expresses in milligrams of malondialdehyde (MDA) equiva-

lents per kilogram of sample, as determined by the methods

described. This assay is derived from the color reaction be-

tween TBA reagent and oxidation products of lipids. Although

most food products naturally contain some level of malondi-

aldehyde as a secondary product of lipid oxidation, especially

affected foods are fried foods, cheese, cooked meats and

dehydrated foods. This assay utilizes the reaction of MDA

with TBA to produce a pink MDATBA adduct, which has an

absorbance maximum at 530540 (532) nm and molar extinc-

tion coefficient at 155 000 M1 cm1 (Kinter, 1995). The forma-

tion of this adduct requires high temperature (90100 C) andacidic conditions. The MDA-TBA adduct so formed can be

measured spectrophotometrically. The TBA assay has been

widely applied in numerous research studies. Close examina-

tion of this method, however, began to divulge that a number

of compounds other than MDA (i.e., alkenals, alkadienals,

browning reaction products, proteins, sugar degradation

products) also react with TBA to form chromogen products,

which absorb at approximately 532 nm. Hence, the results

of this test can be misleading due to lack of specificity. Thus,

the term thiobarbituric acid reactive substances (TBARS) is

currently used instead of TBA (Shahidi & Zhong, 2005). Never-

theless, this is one of the frequently used protocols to detect

oxidative deterioration in lipid-bearing foods. The inhibitory

effect of green tea extract on the oxidation of meat lipids

was reported by Hes, Korczak, and Gramza (2007), who inves-

tigated the effect of this additive on lipid oxidation in minced

pork meat under frozen storage conditions. The authors

determined the content of TBARS in the control sample and

in samples containing 500 ppm tea extract, 500 ppm rose-

mary extract and 200 ppm BHT. After 6-month storage of

samples, the content of TBARS in samples containing rose-

mary and tea extracts were significantly lower than those

found in BHT and control samples. In another experiment

with a meat model system, antioxidant activity of green tea

catechins as evaluated by TBA values was epigallocatechin

gallate epicatechin gallate > epigallocatechin > epicatechin.The inhibitory effect on TBA values of catechins was concen-

Please cite this article in press as: Namal Senanayake, S.P.J., Green tea exview, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.20

tration dependent, being highest at 200 ppm (Shahidi & Alex-

ander, 1998).

8.4. Oil Stability Index (OSI)

The Oil Stability Index (OSI) is an American Oil Chemists

Society approved method (AOCS, 1997; Official Method Cd

12b-92) that determines the relative resistance of fats and oils

to oxidation. Oxidative stability instrument (Omnion Inc.,

Rockland, MA, USA) or Rancimat (Brinkmann Instruments,

Inc., Westbury, NY, USA) can be employed to determine OSI

of oil samples. Both Rancimat and Oxidative Stability Instru-

ment are commercial pieces of equipment for the automated

determination of oxidative resistance and are well-known by

the fats and oils industry. The same basic principle lies be-

hind both the Rancimat and Oxidative Stability Instrument

(Shahidi & Zhong, 2005). The two instruments differ only

slightly in their design and operating conditions. In this

method, a sample of oil or melted fat is weighed into a glass

test tube. The test tube is then placed in a heating block at a

temperature of typically around 110 C. However, the OSI maybe run at temperatures of 80140 C. A stream of purified air ispassed through the sample, and the effluent stream of air is

bubbled through a collection tube containing deionized water.

An electrode is placed in deionized water, and the conductiv-

ity of the water is continually monitored. As the oil oxidizes,

volatile organic acids are produced and trapped in the collec-

tion tube, which increase the conductivity of the water. This

change in conductivity is monitored by computer software.

The Oil Stability Index (OSI) is defined as the point of maxi-

mum change of the rate of oxidation. In a study conducted

by DuPont Nutrition & Health laboratories, the OSI values of

canola oil containing liquid green tea extract were deter-

mined at 110 C. The canola oil sample with nothing addedhad an OSI value of 19.8 h. When liquid green tea extract

(1300 ppm) was added to the canola oil, the OSI value in-

creased up to 31.4 h. Hence, canola oil with green tea extract

found to be more stable as compared to the control sample

with nothing added.

8.5. Oxipres

The Oxipres (Mikrolab Aarhus A/S, Hojbjerg, Denmark), amethod of examining accelerated stability of heterogeneous

food and feed products, is a modification of the traditional

ASTM oxygen bomb method. In this method, the food or feed

samples are placed in a closed container with an oxygen head-

space. Oxidation is accelerated by heating (to 90140 C) and bythe use of oxygen under pressure (initial oxygen pressure of 5

bars). As the product oxidizes, the oxygen content of the head-

space gas decreases which results in a decline of pressure

within the closed container. This reduction in pressure is mea-

sured electronically. Induction period can be determined by

plotting the pressure over time. This induction period (IP) is as-

sumed to be a measure of the resistance of the lipids toward

oxidation. For ease of interpretation, the amount of oxygen

consumed by the sample can be calculated and plotted against

time. Products have a large and fast oxygen consumption (as

indicated by short IP) will be more prone to oxidation, and pre-

sumably have a shorter shelf-life. The Oxipres is normally

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equipped with three thermostatic aluminum heating blocks,

each with two holes allowing six oxygen bombs to be operated

simultaneously at a maximum of three different tempera-

tures. The apparatus may also be equipped with only two oxy-

gen bombs. It is suitable for testing the expected shelf-life of

products with multi-phase systems and systems containing

water (i.e., margarines, fat spreads, mayonnaise, salad dress-

ings, snacks, peanuts, fish meal, chicken meal, meat products,

etc.), which cannot be tested using the OSI. In a study con-

ducted by DuPont Nutrition & Health laboratories, both control

and green tea extract-stabilized fat spreads were made using

standard industry practice. Green tea extract was added at a

dosage level of 200 ppm. Samples were evaluated for Oxipresinduction times at 100 C. Results showed that oxidative stabil-ity of fat spreads was significantly improved using the green

tea extract. Samples supplemented with green tea extract

had higher induction periods as compared to control with

nothing added.

8.6. Schaal oven storage test

The Schaal oven test, also referred to as oven storage test, is

often used for evaluating oils, fats, and foods containing fats

and oils. The AOCS (1997) official method Cg 5-97 describes

the protocol for an accelerated test to measure the stability

of oil by aging in an oven at 60 C. Methods such as peroxidevalue, conjugated dienes, TBARS, sensory evaluation, gas

chromatographic volatile com pounds are then employed to

measure oxidation levels and endpoints in the oils. Because

this test uses relatively low temperatures, the samples are ex-

posed to mild oxidative stress. The results of the Schaal oven

test normally correlate exceptionally well with actual shelf-

life predictions because the end-point represents a lower de-

gree of oxidation (Frankel, 2005). Under Schaal oven test con-

dition at 65 C over a period of 144-h, seal blubber andmenhaden oils treated with tea catechins showed excellent

oxidative stability as compared to samples containing BHA,

BHT, TBHQ, and a-tocopherol. The antioxidant capacity of

catechins in prevention of marine oils oxidation was in the

order of epicatechin gallate > epigallocatechin gallate > epi-

gallocatechin > epicatechin. Moreover, the epigallocatechin

was slightly more effective than TBHQ in the system used

(Wanasundara & Shahidi, 1996).

8.7. Sensory evaluation

Sensory evaluation is a scientific method that is used to mea-

sure, analyze, recall, and interpret the reactions to those char-

acteristics of foods as they are perceived through the human

senses of sight, smell, taste, touch and hearing. Sensory test-

ing requires panels of human participants, on whom the

products are evaluated, and then record the responses made

by them. The sensory quality of a food product is one of the

most crucial aspects influencing its success in the market-

place. All sensory testing protocols are typically categorized

into three broad groups: hedonistic, discriminative and

descriptive testing. Among these, hedonistic tests are always

used within the scope of consumer evaluations and serve to

characterize the consumer behavior. On the other hand, both

discriminative and descriptive sensory tests may only be

Please cite this article in press as: Namal Senanayake, S.P.J., Green tea exview, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.20

carried out by trained/experienced panelists and can give

exceptionally detailed information about individual product

parameters. Hence, the selection of a proper sensory method

is crucial. Sensory evaluation methods are typically expensive

and may require a relatively large number of samples. None-

theless, sensory analyses are typically conducted by the food

industry to evaluate the performance of antioxidants during

shelf-life of food products. Internal studies conducted by Du-

Pont Nutrition & Health have demonstrated that sensory

quality of green tea extract was superior to some commonly

used synthetic antioxidants in chilled (4 C), roasted turkeyburgers. The WOF in the control sample within 24 h of storage

was unusually high. The WOF of samples treated with green

tea extract was not detected even after 13-days of storage at

4 C. Moreover, the sensory attributes of green tea extractwere superior to those of the synthetic antioxidant BHA. In

another study, Mildner-Szkudlarz et al. (2009) showed that

the sensory profile of green tea extract-supplemented biscuits

was superior to samples containing BHA.

9. Conclusions

During the past few decades, the use of natural antioxidants

and plant-derived extracts has received increased interest

due to concerns over possible adverse health effects caused

by the use of synthetic antioxidants. Green tea extract, a nat-

ural source of antioxidant, has been successfully used not

only to enhance flavour but also to extend the shelf-life of

various food products. Green tea extract is particularly suit-

able for products with high susceptibility to lipid oxidation,

including trans-free and low-fat products and foods with a

high content of polyunsaturated fatty acids. The primary

application areas of green tea extract include meat, poultry,

seafood, mayonnaise, salad dressings, soups, sauces, marga-

rines, fat spreads, shortening, frying oils, bakery products,

pizza toppings, cereals and snack foods, among many others.

Research reports on measuring the effectiveness of green tea

extract in food against lipid oxidation are scarce in the litera-

ture. The typical parameters measured in determining the

effectiveness of green tea extract in food include peroxide val-

ues, conjugated dienes, thiobarbituric acid reactive sub-

stances (TBARS), Oil Stability Index (OSI), Oxipres induction

period, and sensory evaluation, among others.

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