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A R T I C L E I N F O A B S T R A C T
Article history: Green tea is one of the most popular and extensively used dietary supplement in the United
Received 20 May 2013 States. Diverse health claims have made for green tea as a trendy ingredient in the growing
Received in revised form market for nutraceuticals and functional foods. Green tea extract contains several polyphe-
16 August 2013 nolic components with antioxidant properties, but the predominant active components are
Accepted 22 August 2013 the flavanol monomers known as catechins, where epigallocatechin-3-gallate and epicate-
Available online 14 September 2013 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
Keywords: epigallocatechin. Among these, epigallocatechin-3-gallate is the most bioactive and the
Green tea extract most scrutinized one. Green tea polyphenols are also responsible for distinctive aroma,
Antioxidant color and taste. Green tea extract can also be used in lipid-bearing foods to delay lipid oxi-
Catechins
dation and to enhance the shelf-life of various food products. This review outlines the
Flavour constituents
chemistry, flavour components, antioxidant mechanism, regulatory status, food applica-
Lipid oxidation
tions, and stability of green tea extract in food.
Food application
Ó 2013 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1530
2. Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1530
3. Flavour constituents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1531
4. Antioxidant mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1532
5. Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1533
6. Method of incorporation into food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534
7. Application in food products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534
8. Stability tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537
8.1. Peroxide value (PV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537
8.2. Conjugated dienes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537
8.3. Thiobarbituric acid (TBA) value. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1538
8.4. Oil Stability Index (OSI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1538
8.5. Oxipresä . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1538
8.6. Schaal oven storage test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1539
1. Introduction 2. Chemistry
Tea, derived from Camellia sinensis L., is one of the most widely The chemical composition of tea leaves has been well docu-
consumed beverages in the world. Tea can be categorized into mented. The main constituents of tea leaves are polyphenols
three main types, depending on the level of oxidation, as (Balentine, Wiseman, & Bouwens, 1997). The fresh tea leaves
green tea, oolong tea and black tea (Chan, Soh, Tie, & Law, contain caffeine (approximately 3.5% of the total dry weight),
2011; Velayutham, Babu, & Liu, 2008). Green tea is an ever- theobromine (0.15–0.2%), theophylline (0.02–0.04%) and other
green plant that grows primarily in tropical and temperate re- methylxanthines, lignin (6.5%), organic acids (1.5%), chloro-
gions of Asia which mainly include China, India, Sri Lanka phyll (0.5%) and other pigments, theanine (4%) and free amino
and Japan. It is also cultivated in several African and South- acids (1–5.5%), and numerous flavour compounds (Graham,
American countries. Two primary varieties of C. sinensis are 1992). In addition, a wide variety of other components exists,
Camellia sinensis sinensis and Camellia sinensis assamica. The including, flavones, phenolic acids and depsides, carbohy-
sinensis plant strain is originated from China. This strain pro- drates, alkaloids, minerals, vitamins and enzymes (Chaturve-
duces green, white, black and oolong teas. On the contrary, dula & Prakash, 2011). Tea also contains flavonols, mainly
the assamica plant strain primarily is inhabitant to the Assam quercetin, kaempferol, myricetin, and their glycosides. The
region in Northern India. Due to enormous yields of this spe- most favorable effects of green tea are accredited to the green
cific strain, it is the favored plant grown in India, Sri Lanka tea polyphenols, predominantly the catechins, which make
and some African countries. The leaves of assamica strain up, 25–35% of the dry weight of green tea leaves (Abdel-Rah-
are typically used for producing black, oolong and pu’erh teas. man et al., 2011; Balentine et al., 1997; Graham, 1992; Zaveri,
Green tea is a small shrub that can expand up to 30 feet high, 2006). The tea catechins belong to the family of flavonoids
but is customarily trimmed to 2–5 feet when cultivated for its (Yilmaz, 2006) and possess two benzene rings referred to as
leaves. The leaves are naturally murky green and glossy with the A- and B-rings. In addition, the catechin molecules con-
notched edges and are 2–5 cm broad and 4–15 cm long. The tain a dihydropyran heterocycle (the C-ring) that has a hydro-
flowers are white and contain bright yellow stamens. These xyl group on carbon 3. Moreover, the A-ring is similar to a
blossoms appear individually or as clusters. The fruits have resorcinol moiety whereas the B-ring is similar to a catechol
hard green shells with round, brown-colored seeds. These moiety. The catechin molecule has two chiral centers on car-
seeds can be used to produce tea oil. Typically, flowering is bons 2 and 3. Hence, it has four diastereoisomers with two of
prevented during cultivation by harvesting the leaves. The the isomers are in trans configuration, and the other two are
immature, light-green leaves are preferably harvested for in cis configuration. The trans and cis isomers are referred to
tea production. Mature leaves are deeper green in color than as the catechin and epicatechin, respectively. These chemical
the young leaves. Different leaf ages produce varying tea structures appear to be important for the antioxidant activi-
qualities as their chemical compositions are different. Typi- ties of tea polyphenols, including an ortho-3 0 4 0 -dihydroxyl
cally, the buds (tips) and the first two to three leaves are har- group or 3 0 4 0 5 0 -trihydroxyl group in the B-ring, a gallate group
vested by hand picking for processing. This process is located at the 3 position of the C-ring, and hydroxyl groups at
generally repeated every one to two weeks. Green, white, the 5 and 7 positions of the A-ring (Rice-Evans, Miller, & Pa-
black and oolong teas all originate from the leaves of C. sinen- ganga, 1996). Many structure–activity relationship investiga-
sis plant. However, the different types of tea are classified tions have been performed on the antioxidant activity of
according to the degree of fermentation that takes place dur- flavonoids, including tea catechins (Farkas, Jakus, & Héberger,
ing processing: both white tea and green tea being unfer- 2004; Guo et al., 1999; Harborne & Williams, 2000; Heim,
mented; oolong tea semi-fermented and black tea fully Tagliaferro, & Bobilya, 2002; Rice-Evans et al., 1996). According
fermented (Chan et al., 2011). These basic types of tea have to these studies, the antioxidant activity of flavonoids de-
different quality characteristics, including appearance, aro- pends substantially on the number and position of hydroxyl
ma, taste, and color. The manufacturing process of tea is groups in the molecule (Farkas et al., 2004). In addition, sev-
designed to either preclude, or permit tea polyphenolic com- eral structural elements such as o-dihydroxyl catechol struc-
pounds to be oxidized by naturally occurring polyphenol ture in the B-ring, the presence of unsaturation and 4-oxo
oxidase in the tea leaves. Green tea is produced by inactivat- group in the C-ring are also presumed to increase the antiox-
ing the heat-labile enzyme polyphenol oxidase in the fresh idant activity of flavonoids. The 2,3-double bond in the C-ring
leaves by either applying heat or steam, which prevents the along with 4-oxo function in the C-ring facilitates electron
enzymatic oxidation of catechins, the most abundant delocalization from the B-ring. Moreover, hydroxyl groups at
flavonoid compounds present in green tea extracts (Velayu- positions 3 and 5 providing hydrogen bonding to the 4-oxo
tham et al., 2008). The tea leaves are then rolled, dried and group in the C-ring is another structural feature attributed
packaged. to the antioxidant activity of flavonoids.
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 15 2 9–15 4 1 1531
hydroperoxides (LOOH) by the hemolytic cleavage of the O–O activities of catechin, epicatechin and epicatechin gallate
bond and producing lipid alkoxyl radicals. Phenolic antioxi- were ten times higher than those of L-ascorbate and beta-car-
dants, including tea catechins, inhibit lipid peroxidation by otene. In another study, Nanjo et al. (1996) documented that
binding these lipid alkoxyl radicals. This activity depends on DPPH radical scavenging activities of catechin and epicate-
the structure of the molecules, and the number and position chin are less than epigallocatechin, epicatechin gallate, and
of the hydroxyl group in the molecules (Milić, Djilas, & Cana- epigallocatechin gallate. As expected, epicatechin is another
danovic-Brunet, 1998). Green tea catechins also exhibit anti- monomeric flavanol (Yilmaz, 2006) came across in green tea.
oxidant activity through inhibiting pro-oxidant enzymes Reports have shown that epicatechin is capable of scavenging
and inducing antioxidant enzymes (Velayutham et al., 2008). hydroxyl radicals, peroxyl radicals, superoxide radicals, and
The antioxidant activity of these catechins and the deriv- DPPH radicals (Bors & Michel, 1999; Fukumoto & Mazza,
atives showed a marked difference depending on the sub- 2000; Liu, Ma, Zhou, Yang, & Liu, 2000; Yilmaz, 2006). Yashin,
strate used for evaluation (Wanasundara & Shahidi, 2005). Yashin, and Nemzer (2011) examined the antioxidant activity
Green tea catechins, which performed like other hydrophilic of different types of teas that include green, oolong, black and
antioxidants such as Trolox (a water-soluble analog of a- pu-erh teas. The antioxidant activity of teas descends in the
tocopherol) and ascorbic acid, have been shown to be active following order: green > oolong > black > pu’erh tea. Chan
antioxidants in bulk oils, but were prooxidants in the corre- et al. (2011) evaluated the role of non-polymeric phenolic
sponding oil-in-water emulsions (Frankel, Huang, & Aesc- (NP) and polymeric tannin (PT) constituents in the antioxi-
bach, 1997; Frankel, Huang, Kanner, & German, 1994). In dant and antibacterial properties of six brands of green, black,
corn oil triacylglycerols system that was oxidized at 50 °C and herbal teas. Extraction yields of the products ranged from
the antioxidant activities of epigallocatechin, epigallocate- 12% to 23%. Much higher fractionation yields more NP con-
chin gallate and epicatechin gallate were superior to those stituents (70–81%) than PT constituents (1–11%), which sug-
of epicatechin or catechin (Huang & Frankel, 1997). These cat- gested that, the former are the principal tea components. In
echins have also been highly successful in delaying oxidation general, the antioxidant properties of green tea extracts were
of polyunsaturated fatty acids-rich marine and vegetable oils stronger than those of black and herbal teas (Chan et al.,
(Wanasundara & Shahidi, 1998). In the lecithin-containing lip- 2011). For all six brands tested, antioxidant properties of PT
osomes system, epigallocatechin gallate was superior to other fractions were significantly higher than crude extracts and
compounds such as epicatechin, epigallocatechin, epicate- NP fractions. Although PT constituents have stronger antiox-
chin gallate, and catechin (Huang & Frankel, 1997). In the idant and antibacterial properties, they constitute only a min-
oil-in-water emulsions, however, all catechin compounds or component of the teas (Chan et al., 2011).
demonstrated pro-oxidant activity (Huang & Frankel, 1997). Green tea polyphenols have been shown to be effective
The improved antioxidant activity observed for tea catechins against beta-carotene oxidation. For example, tea catechins
in liposomes compared to emulsions has been explained by have been able to demonstrate an anti-discoloring effect on
the greater affinity of the polar catechins toward the polar beverages and margarine containing beta-carotene (Koketsu,
surface of the lecithin bilayers, thus affording better protec- 1997; Unten, Koketsu, & Kim, 1997). It was suggested that
tion (Huang & Frankel, 1997). In a study conducted by Zhong the discoloration of beta-carotene and the oxidation of unsat-
and Shahidi (2011), epigallocatechin gallate was structurally urated fatty acids progressed by the same mechanism. In par-
modified to improve its lipophilicity via esterification of epi- ticular, carbon–carbon double bonds in beta-carotene and
gallocatechin gallate with selected fatty acids, such as stearic unsaturated fatty acids are attacked by radicals, becoming
acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid hydroperoxides. Green tea catechins have the ability to sup-
(DHA). The lipophilized derivatives so produced exhibited press the degradation of double bonds as radical scavengers.
greater antioxidant activity than the original epigallocatechin It was also suggested that the hydroxyl group at the 5 0 -posi-
gallate molecule. tion of the B-ring of the catechin structure mostly contributed
Several investigators have speculated the mechanism of to the anti-discoloring effect. Hence, as described above, it
antioxidant action of (+)-catechin using some oxidation mod- was speculated that green tea polyphenols delayed the degra-
el studies (Hirose, Yamamoto, & Nakayama, 1990; Koketsu, dation of beta-carotene by acting as antioxidants following
1997; Zhu et al., 2000). As indicated by the proposed mecha- the same mechanism. Among the individual green tea poly-
nism, (+)-catechin molecule can scavenge four lipid free rad- phenols examined, gallocatechin gallate, epigallocatechin
icals per molecule (Hirose et al., 1990; Koketsu, 1997). The gallate, epigallocatechin, and gallocatechin showed strong
antioxidant activity of individual tea polyphenols in different anti-discoloring effect while epicatechin and catechin
model systems showed a proportional relationship to the showed almost no activity, and gallic acid had moderate
number of hydrogen radical donors of catechins. A synergistic activity (Unten et al., 1997).
effect has been observed between green tea catechins and
ascorbic acid and a-tocopherols (Murakami, Yamaguchi, 5. Regulatory status
Takamura, & Matoba, 2003). Catechin, a monomeric flavanol,
is reported to have hydroxyl, peroxyl, superoxide and 1,1-di- In the United States, green tea extract may be used as flavour
phenyl-2-picrylhydrazyl (DPPH) radical scavenging activities agent with antioxidative properties in various fats, oils and
(Bors & Michel, 1999; Fukumoto & Mazza, 2000). Moreover, foods containing fats and oils. Green tea has a history of safe
tea catechins have the ability to chelate iron in food model use as food flavouring and is known for its antioxidative func-
systems (Tang, Kerry, Sheehan, & Buckley, 2002). Nakao, Taki- tionality as a secondary effect. Green tea extract is well
o, and Ono (1998) reported that the peroxyl radical scavenging known in Asian food. According to the United States Code
1534 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 5 2 9 –1 5 4 1
of Federal Regulations (21 CFR 101.22, and 21 CFR 182.10), the ferred in certain formulations containing dry ingredients as
term ‘natural flavour’ or ‘natural flavouring’ means the essen- this enables easier blending. The powder carrier aids the
tial oil, oleoresin, essence or extractive, protein hydrolysate, incorporation and homogeneous distribution of the green
distillate, or any product of roasting, heating or enzymolysis, tea extract. Where dry ingredients are part of the formulation,
which contains the flavouring constituents derived from a a powdered green tea extract is often preferred due to easier
spice, fruit or fruit juice, vegetable or vegetable juice, edible blending. Conversely, Green tea extract can be dissolved in a
yeast, herb, bark, bud, root, leaf or similar plant material, food-grade solvent to produce an oil soluble or dispersible li-
meat, seafood, poultry, eggs, dairy products or fermentation quid product. Liquid forms of green tea extracts may be added
products thereof, whose significant function in food is fla- to food products by direct addition into oils and fats. The fat
vouring rather than nutritional (U.S. Food and Drug Adminis- or oil may be heated to an elevated temperature (i.e., 40–
tration, 2012). Natural flavours include the natural essence or 60 °C) while stirring. Liquid green tea extract should then be
extractives obtained from plants listed in sections CFR 182.10, added slowly into melted fat or oil. After addition is com-
182.20, 182.40, and 182.50 and part 184 of this chapter, and the pleted, agitation should be continued for an additional period
substances listed in section 172.510 of this chapter (U.S. Food of time for complete and uniform distribution of green tea ex-
and Drug Administration, 2012). Thus, in the United States, tract in fats and oils.
green tea extract may be labeled as natural flavouring. In
European Union (EU), the definition of natural flavouring sub- 7. Application in food products
stances, as stated in Council Directive 88/388/EEC, applies to
green tea extracts when used as flavourings, and they can There is growing consumer interest in the green tea extract-
be used in all food applications, unless regulations restrict supplemented products in recent years. Green tea extract
the use of flavourings in specific foods (Official Journal of has been used in a variety of food products including bread
the European Union, 2008). Certain green tea extracts are also (Wang & Zhou, 2004), biscuits, dehydrated apple (Lavelli, Van-
regarded as traditional foods in the EU, however, market taggi, Corey, & Kerr, 2010) and various meat products (Mit-
clearance of green tea extracts depends on the intended use sumoto, O’Grady, Kerry, & Buckley, 2005; O’Sullivan, Lynch,
as well as the product profile. If the primary function high- Lynch, Buckley, & Kerry, 2004; Tang, Kerry, Sheehan, & Buck-
lighted is the antioxidant properties, the green tea extract is ley, 2001). The main application areas of green tea extract
then regulated as an antioxidant and must be specifically ap- are summarized in Table 1. In internal tests with fat spreads,
proved. Results of various investigations have confirmed that green tea extract showed comparable antioxidant perfor-
green tea polyphenolic compounds are largely responsible for mance to conventional synthetic antioxidant tert-butylhydro-
distinctive aroma, color and taste (Borse et al., 2002; Chat- quinone (TBHQ) and may be more cost-effective than other
urvedula & Prakash, 2011; Pripdeevech & Machan, 2011; natural sources of antioxidants. This makes it ideal for both
Yamanishi, 1995). Hence, various compounds of green tea ex- full-fat margarines as well as oxidation-sensitive products
tract that contribute to the antioxidant effects also contribute that are low in fat, free of trans fatty acids or have a high con-
to flavour properties. The combination of these functional- tent of polyunsaturated fatty acids. In this study, samples of
ities may allow innovative solutions such as designing spe- 40% low-fat spread were produced with green tea extract con-
cific green tea extract products to target certain food taining 60 ppm total catechins or 100 ppm TBHQ and stored at
applications. As green tea extract contributes to the flavour 5 °C for 13 weeks. A control sample contained no antioxidant
profile of food matrix and helps retard lipid oxidation, it can solution. In an accelerated shelf-life test, a comparison of
be successfully used in a wide array of oils, fats, and foods green tea extract and TBHQ revealed similar performance.
containing fats and oils. Lipid oxidation is one of the primary causes of quality
deterioration in meat and poultry products. Red meats and
6. Method of incorporation into food poultry are typically highly susceptible to lipid oxidation
due to their high fat content (Yilmaz, 2006) and a high propor-
Proper incorporation of green tea extract into food products is tion of polyunsaturated fatty acids. These highly unsaturated
imperative to ensure that antioxidant components are thor- phospholipids present in meat and poultry products are
oughly dissolved and/or homogeneously dispersed in the responsible for the development of off-flavours and off-odors
food matrix. As only a small amount of green tea extract after cooking and subsequent refrigerated storage. This off-
may be required for adequate shelf-life extension in food, flavour development, also referred to as warmed-over-flavour
the method of application may determine the achievement (WOF), frequently causes consumers to reject freshly cooked
of the antioxidant effectiveness. The selection of application meat and poultry products. Therefore, these products should
methods may depend on the type of food products, process- be protected by natural sources of antioxidants such as green
ing methods, and the availability of the equipment used for tea extract to protect against WOF.
food processing. Commercial extracts of green tea are mainly In a study conducted by DuPont Nutrition & Health labora-
available in a fine-grained powder form. Green tea extract can tories, both control and green tea extract-stabilized turkey
be solubilized in water prior to addition into foods. Water-sol- burgers were made using standard industry practice. Green
uble green tea extract has a low viscosity for easy spraying tea extract was added, at a dosage level of 50–100 ppm. Sam-
and homogeneous distribution. Moreover, remarkably little ples were evaluated for thiobarbituric acid reactive sub-
energy is required to produce the dispersion in water. On stances (TBARS) formation, reported as malondialdehyde (a
the other hand, green tea extract in powder form is often pre- secondary product of lipid oxidation) equivalents. Results
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 15 2 9–15 4 1 1535
Fig. 2 – TBARS of roasted turkey burgers during storage at Fig. 3 – Effect of green tea extract and BHA on the inhibition
4 °C. of TBARS of roasted turkey burgers during storage at 4 °C.
Fig. 5 – Color of raw ground beef patties treated with green tea extract and rosemary extract compared to an unprotected
control sample.
1000 ppm), sage extract (at 1000 ppm), or tea catechins (at extract. In addition, green tea extract contributed to ‘‘meaty’’
100 ppm). Their results indicated that the addition of natural (umami) taste in finished burgers (data not shown). In flavour
extracts, including tea catechins, reduced lipid oxidation in sensitive meat systems, green tea extract would be a more
chicken nuggets both in the presence and absence of salt. viable option than the rosemary extract due to similar effec-
However, when sodium tripolyphosphate (STPP) was incorpo- tiveness, but lower flavour impact. A study by Moawad, Abo-
rated into the same product system, the efficacy of natural ex- zeid, and Nadir (2012) has shown that the use of tea catechins
tracts was significantly reduced. in combination with nitrite was more effective in keeping
In addition to providing antioxidant activity and natural quality of dry-cured sausages during refrigerated storage as
flavor attributes, green tea extract can also help improve the indicated by attractive red color (a* value) as compared to
color stability of fresh meat products. Fresh meat color de- samples treated with nitrite. In another study conducted by
pends on myoglobin that stores oxygen for aerobic metabo- DuPont Nutrition & Health laboratories, cured ham slices con-
lism in the muscle. Iron is a pivotal player in meat color. taining 500 ppm sodium ascorbate and 300 ppm green tea ex-
One of the defining factors of meat color is the chemical state tract enhanced color stability (from 14 days to 25 days) by
of iron. Oxymyoglobin gives meats the red color. The brown- maintaining the redness remarkably efficiently as compared
ish color is due to metmyoglobin formed by oxidation of oxy- to a standard ham with 500 ppm sodium ascorbate. The
myoglobin. However, how green tea extract influences the ham slices were packaged under MAP conditions (80% oxygen
color cycle in meat products is not well understood. Studies and 20% carbon dioxide) and stored at 5 °C for a period of
conducted by DuPont Nutrition & Health have shown that 38 days. This was shown visually and measured by a Videom-
the red color of raw ground beef patties (20% fat) was signifi- eterLab 2 – High Performance Multispectral Imaging by
cantly improved by the addition of rosemary extract recording a-values. The a-value of 17 was defined as the work-
(1000 ppm) and green tea extract (250 ppm) compared to the ing limit for redness acceptability of cured ham.
development of browning in control samples (Fig. 5). All sam- Results of numerous investigations have also confirmed the
ples were stored at 0–2 °C for 22 days and packaged under antioxidant properties of green tea catechins in various lipid
modified atmosphere (80% oxygen and 20% carbon dioxide) and food model systems (Amarowicz & Shahidi, 1995; Chen,
packaging conditions. Determination of redness of the meat Chan, Ma, Fung, & Wang, 1996; Frankel et al., 1997; Huang &
(‘a’ value using Minolta Colorimeter) corresponded with the Frankel, 1997; Koketsu & Satoh, 1997; Matsuzaki & Hara,
development as the visible red color. The meat had lost 1985; Mildner-Szkudlarz, Zawirska-Wojtasiak, Obuchowski, &
almost if not all visible red color when the ‘a’ value reached Goslinski, 2009; Roedig-Penman & Gordon, 1997; Wanasundara
approximately 12. The control reached this point around & Shahidi, 1998). When green tea catechins were added to noo-
day 9, and the treated samples around day 16. This shows dles and to the edible oils (soybean, fish, lard) they were able to
that 1000 ppm rosemary extract and 250 ppm green tea improve the oxidative stability of the fried product and the oil
extract exhibited good protection of color for several days be- used for frying (Frankel et al., 1997; Koketsu & Satoh, 1997). In a
yond the control sample, and there was a minimal difference study with marine oils such as seal blubber oil and menhaden
between the two treatments (Fig. 5). Sensory evaluation of the oil, both antioxidant and pro-oxidant effects of green tea ex-
burgers showed that green tea extract had no unfavorable fla- tracts have been reported (Wanasundara & Shahidi, 1998).
vour impact on the finished burgers where slight rosemary Green tea extracts demonstrated a pro-oxidant effect in both
flavour could be detected in the samples containing rosemary oils evaluated, possibly due to the presence of their chlorophyll
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 15 2 9–15 4 1 1537
components. Consequently, in follow-up studies, a column use conditions and examining them periodically for organo-
chromatography was utilized to eliminate chlorophyll com- leptic (odor and flavour) changes or by testing them chemically
pounds from green tea extracts. The dechlorophyllized green for oxidative rancidity. Although normal use conditions are
tea extract so produced was incorporated into both seal blub- ideal, this protocol may be too slow to be of practical value.
ber and menhaden oils at various levels. The antioxidant activ- Current methods for determining the effectiveness of green
ity of dechlorophyllized green tea extract was compared with tea extract against lipid oxidation in food are discussed in this
that of the butylated hydroxyanisole (BHA), butylated section.
hydroxytoluene (BHT), TBHQ and a-tocopherol. At the levels
of P200 ppm, dechlorophyllized green tea extract showed 8.1. Peroxide value (PV)
exceptional antioxidant activity in both oils examined. More-
over, the efficacy of dechlorophyllized green tea extract was The peroxide value (PV), an indicator of primary products of
superior to that of the BHA (at 200 ppm), BHT (at 200 ppm) lipid oxidation, has been the most commonly employed
and a-tocopherol (at 500 ppm), but inferior to that of TBHQ chemical assay for evaluating oxidative stability of fats and
(at 200 ppm). In several other studies, antioxidant activity of oils. In this test, the melted fat or oil sample is titrated with
tea polyphenols in vegetable oils and animal fats was exam- a mixture of glacial acetic acid–chloroform (3:2, v/v) and sat-
ined (Chen et al., 1996; Koketsu, 1997; Koketsu & Satoh, urated potassium iodide (KI) solution. As hydroperoxides
1997). Tea polyphenols evidently demonstrated an inhibitory present in the sample oxidize iodide to iodine, the liberated
effect on oxidation of lard, and this effect was dose-depen- iodine is then titrated with standardized solution of sodium
dent. Furthermore, there was a distinct suppressive effect of thiosulfate to an end-point. This method is highly empirical,
tea polyphenols on oxidative rancidity of soybean oil (Koketsu, and any discrepancy in the test procedure may result in a var-
1997). When green tea extract was compared with BHA and iation of results. The PV is calculated by determining all sub-
tocopherols, green tea catechins showed better antioxidant stances which oxidize KI, in terms of milli-equivalents of
activity in fish oil (Koketsu & Satoh, 1997). The ethanol extracts peroxides per kilogram of sample (Shahidi & Zhong, 2005).
of green tea catechins strongly inhibited the oxidation of cano- These substances are generally assumed to be peroxides or
la oil at 100 °C as shown by measurement of oxygen consump- other similar products of oil oxidation. The content of hydro-
tion, whereas extracts of black tea showed little to no peroxides can be calculated directly by titration of iodine so
antioxidant activity (Chen et al., 1996). Crude green tea extract produced. In order to avoid the use of chloroform, the Amer-
powders were more effective than a-tocopherol and BHA in ican Oil Chemists Society (AOCS, 2003) has developed an
lard under conditions of the active oxygen method (AOM) at alternative method, which uses isooctane as solvent (AOCS,
97.8 °C (Matsuzaki & Hara, 1985). In a b-carotene-linoleate 2003; Official Method Cd 8b-90), although the method is lim-
model system, ()-epicatechin-3-gallate exhibited the stron- ited to PV of less than 70 meq/kg as described in the AOCS
gest antioxidative activity whereas ()-epigallocatechin had guidelines. The results of this test are linked to flavor precur-
the weakest performance (Amarowicz & Shahidi, 1995). Fur- sors such as hydroperoxides, but not to the actual flavour
thermore, the antioxidative efficacy of ()-epicatechin and compounds. In an internal study, samples of 40% low-fat
()-epigallocatechin-3-gallate was similar and in between spreads were produced with green tea extract containing
those of ()-epicatechin-3-gallate and ()-epigallocatechin. 60 ppm total catechins or 100 ppm TBHQ and stored at 5 °C
In another study, Mildner-Szkudlarz et al. (2009) examined for 13 weeks. A control sample contained no antioxidant solu-
the feasibility of using green tea extract to improve the oxida- tion. While the hydroperoxides content has significantly in-
tive stability of biscuits during storage. The antioxidant activ- creased in the control sample, the samples containing green
ity of green tea extract was compared with commonly used tea extract or TBHQ had significantly fewer hydroperoxides
synthetic antioxidant BHA. Phenolic compounds of green tea after 15 weeks of storage. In another study, Mustafa (2013)
extract had significantly higher DPPH radical scavenging activ- demonstrated that green tea extract inhibited lipid hydroper-
ity than BHA. However, both green tea extract and BHA were oxides formation in ground beef during refrigerated storage
effective inhibitors of the decomposition of hydroperoxides. (Mustafa, 2013).
Moreover, biscuits treated with green tea extract had better
sensory profile during storage as compared to samples con- 8.2. Conjugated dienes
taining BHA.
Oxidation of highly unsaturated fatty acids is associated with
an increase in the ultra violet (UV) absorption of the product.
8. Stability tests During lipid oxidation, double bonds in unsaturated fatty
acids are changed from non-conjugated to conjugated double
Oxidative stability is a decisive factor in the processing and bonds. This process takes place almost immediately after
marketing of oils, fats, and lipid-bearing foods. Methods for hydroperoxides have been formed (Gunstone & Norris,
determining oxidative stability are, therefore, essential partic- 1983). These conjugated diene hydroperoxides have a UV
ularly when green tea extract is being evaluated for its effec- maximum at 230–240 nm, with a strong characteristic absorp-
tiveness in delaying lipid oxidation in these products. tion at 232 nm. Formation of conjugated dienes is an indicator
Consequently, many protocols have been developed in at- of detection of primary changes in lipid oxidation. Compared
tempts to assess the effectiveness of green tea extract in food. to peroxide value determination, the measurement of conju-
Oxidative stability of green tea extract-supplemented food gated dienes is much simpler and faster and does not rely on
products may be determined by storing samples at normal chemical reactions (Shahidi & Zhong, 2005) or formation of
1538 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 5 2 9 –1 5 4 1
color development. The inhibition of formation of conjugated gallate epicatechin gallate > epigallocatechin > epicatechin.
dienes by green tea extract or other sources of antioxidants The inhibitory effect on TBA values of catechins was concen-
can be used as means of assessing antioxidant performance tration dependent, being highest at 200 ppm (Shahidi & Alex-
in food. Formation of conjugated dienes is reported to corre- ander, 1998).
late well with peroxide values during the early stages of lipid
oxidation (Wanasundara, Shahidi, & Jablonski, 1995). During 8.4. Oil Stability Index (OSI)
Internal testing on 40% fat spreads, the addition of 300 ppm
green tea extract was adequate to inhibit conjugated dienes The Oil Stability Index (OSI) is an American Oil Chemists’
formation during 13 weeks of storage at 5 °C. In another Society approved method (AOCS, 1997; Official Method Cd
internal study, samples of 40% low-fat spreads were prepared 12b-92) that determines the relative resistance of fats and oils
with green tea extracts containing 40 ppm catechins, and to oxidation. Oxidative stability instrument (Omnion Inc.,
60 ppm catechins or 100 ppm TBHQ solution and stored at Rockland, MA, USA) or Rancimat (Brinkmann Instruments,
5 °C for 15 weeks. The addition of green tea extract had a Inc., Westbury, NY, USA) can be employed to determine OSI
dose-dependent effect on the inhibition of conjugated dienes. of oil samples. Both Rancimat and Oxidative Stability Instru-
Moreover, the addition of green tea extract with 60 ppm ment are commercial pieces of equipment for the automated
catechins and 100 ppm TBHQ inhibited the development of determination of oxidative resistance and are well-known by
conjugated dienes most effectively throughout the storage the fats and oils industry. The same basic principle lies be-
period. hind both the Rancimat and Oxidative Stability Instrument
(Shahidi & Zhong, 2005). The two instruments differ only
8.3. Thiobarbituric acid (TBA) value slightly in their design and operating conditions. In this
method, a sample of oil or melted fat is weighed into a glass
TBA is one of the most widely used methods of assessing the test tube. The test tube is then placed in a heating block at a
extent of lipid oxidation in foods. The TBA value is typically temperature of typically around 110 °C. However, the OSI may
expresses in milligrams of malondialdehyde (MDA) equiva- be run at temperatures of 80–140 °C. A stream of purified air is
lents per kilogram of sample, as determined by the methods passed through the sample, and the effluent stream of air is
described. This assay is derived from the color reaction be- bubbled through a collection tube containing deionized water.
tween TBA reagent and oxidation products of lipids. Although An electrode is placed in deionized water, and the conductiv-
most food products naturally contain some level of malondi- ity of the water is continually monitored. As the oil oxidizes,
aldehyde as a secondary product of lipid oxidation, especially volatile organic acids are produced and trapped in the collec-
affected foods are fried foods, cheese, cooked meats and tion tube, which increase the conductivity of the water. This
dehydrated foods. This assay utilizes the reaction of MDA change in conductivity is monitored by computer software.
with TBA to produce a pink MDA–TBA adduct, which has an The Oil Stability Index (OSI) is defined as the point of maxi-
absorbance maximum at 530–540 (532) nm and molar extinc- mum change of the rate of oxidation. In a study conducted
tion coefficient at 155 000 M1 cm1 (Kinter, 1995). The forma- by DuPont Nutrition & Health laboratories, the OSI values of
tion of this adduct requires high temperature (90–100 °C) and canola oil containing liquid green tea extract were deter-
acidic conditions. The MDA-TBA adduct so formed can be mined at 110 °C. The canola oil sample with nothing added
measured spectrophotometrically. The TBA assay has been had an OSI value of 19.8 h. When liquid green tea extract
widely applied in numerous research studies. Close examina- (1300 ppm) was added to the canola oil, the OSI value in-
tion of this method, however, began to divulge that a number creased up to 31.4 h. Hence, canola oil with green tea extract
of compounds other than MDA (i.e., alkenals, alkadienals, found to be more stable as compared to the control sample
browning reaction products, proteins, sugar degradation with nothing added.
products) also react with TBA to form chromogen products,
which absorb at approximately 532 nm. Hence, the results 8.5. Oxipresä
of this test can be misleading due to lack of specificity. Thus,
the term ‘‘thiobarbituric acid reactive substances’’ (TBARS) is The Oxipresä (Mikrolab Aarhus A/S, Hojbjerg, Denmark), a
currently used instead of TBA (Shahidi & Zhong, 2005). Never- method of examining accelerated stability of heterogeneous
theless, this is one of the frequently used protocols to detect food and feed products, is a modification of the traditional
oxidative deterioration in lipid-bearing foods. The inhibitory ASTM oxygen bomb method. In this method, the food or feed
effect of green tea extract on the oxidation of meat lipids samples are placed in a closed container with an oxygen head-
was reported by He˛ś, Korczak, and Gramza (2007), who inves- space. Oxidation is accelerated by heating (to 90–140 °C) and by
tigated the effect of this additive on lipid oxidation in minced the use of oxygen under pressure (initial oxygen pressure of 5
pork meat under frozen storage conditions. The authors bars). As the product oxidizes, the oxygen content of the head-
determined the content of TBARS in the control sample and space gas decreases which results in a decline of pressure
in samples containing 500 ppm tea extract, 500 ppm rose- within the closed container. This reduction in pressure is mea-
mary extract and 200 ppm BHT. After 6-month storage of sured electronically. Induction period can be determined by
samples, the content of TBARS in samples containing rose- plotting the pressure over time. This induction period (IP) is as-
mary and tea extracts were significantly lower than those sumed to be a measure of the resistance of the lipids toward
found in BHT and control samples. In another experiment oxidation. For ease of interpretation, the amount of oxygen
with a meat model system, antioxidant activity of green tea consumed by the sample can be calculated and plotted against
catechins as evaluated by TBA values was epigallocatechin time. Products have a large and fast oxygen consumption (as
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 15 2 9–15 4 1 1539
indicated by short IP) will be more prone to oxidation, and pre- used within the scope of consumer evaluations and serve to
sumably have a shorter shelf-life. The Oxipresä is normally characterize the consumer behavior. On the other hand, both
equipped with three thermostatic aluminum heating blocks, discriminative and descriptive sensory tests may only be
each with two holes allowing six oxygen bombs to be operated carried out by trained/experienced panelists and can give
simultaneously at a maximum of three different tempera- exceptionally detailed information about individual product
tures. The apparatus may also be equipped with only two oxy- parameters. Hence, the selection of a proper sensory method
gen bombs. It is suitable for testing the expected shelf-life of is crucial. Sensory evaluation methods are typically expensive
products with multi-phase systems and systems containing and may require a relatively large number of samples. None-
water (i.e., margarines, fat spreads, mayonnaise, salad dress- theless, sensory analyses are typically conducted by the food
ings, snacks, peanuts, fish meal, chicken meal, meat products, industry to evaluate the performance of antioxidants during
etc.), which cannot be tested using the OSI. In a study con- shelf-life of food products. Internal studies conducted by Du-
ducted by DuPont Nutrition & Health laboratories, both control Pont Nutrition & Health have demonstrated that sensory
and green tea extract-stabilized fat spreads were made using quality of green tea extract was superior to some commonly
standard industry practice. Green tea extract was added at a used synthetic antioxidants in chilled (4 °C), roasted turkey
dosage level of 200 ppm. Samples were evaluated for Oxipresä burgers. The WOF in the control sample within 24 h of storage
induction times at 100 °C. Results showed that oxidative stabil- was unusually high. The WOF of samples treated with green
ity of fat spreads was significantly improved using the green tea extract was not detected even after 13-days of storage at
tea extract. Samples supplemented with green tea extract 4 °C. Moreover, the sensory attributes of green tea extract
had higher induction periods as compared to control with were superior to those of the synthetic antioxidant BHA. In
nothing added. another study, Mildner-Szkudlarz et al. (2009) showed that
the sensory profile of green tea extract-supplemented biscuits
was superior to samples containing BHA.
8.6. Schaal oven storage test
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