Food Chemistry 127 (2011) 589–595

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Stability and anti-glycation properties of intermediate moisture apple products

fortified with green tea
Vera Lavelli a,⇑, Mark Corey b, William Kerr b, Claudia Vantaggi a
a
DISTAM, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
b
Department of Food Science and Technology, University of Georgia, Food Science and Technology Building, 100 Cedar St., Athens, GA 30602, United States

a r t i c l e i n f o a b s t r a c t

Article history: Intermediate moisture products made from blanched apple flesh and green tea extract (about 6 mg of
Received 1 June 2010 monomeric flavan 3-ols added per g of dry apple) or blanched apple flesh (control) were produced,
Received in revised form 2 December 2010 and their quality attributes were investigated over storage for two months at water activity (aw) levels
Accepted 12 January 2011
of 0.55 and 0.75, at 30 °C. Products were evaluated for colour (L⁄, a⁄, and b⁄ Hunter’s parameters), phyto-
Available online 18 January 2011
chemical contents (flavan 3-ols, chlorogenic acid, dihydrochalcones, ascorbic acid and total polyphenols),
ferric reducing antioxidant potential, 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl radical-scavenging
Keywords:
activity and ability to inhibit formation of fructose-induced advanced glycation end-products.
Intermediate moisture foods
Apple
During storage of the fortified and unfortified intermediate moisture apples, water availability was suf-
Green tea ficient to support various chemical reactions involving phytochemicals, which degraded at different
Anti-glycation activity rates: ascorbic acid > flavan 3-ols > dihydrochalcones and chlorogenic acid. Colour variations occurred
Radical scavenging activity at slightly slower rates after green tea addition. In the intermediate moisture apple, antioxidant and
anti-glycoxidative properties decreased at similar rates (half-life was about 80 d at aw of 0.75, 30 °C).
In the green tea-fortified intermediate moisture apple, the antioxidant activity decreased at a slow rate
(half-life was 165 d at aw of 0.75, 30 °C) and the anti-glycoxidative properties did not change, indicating
that flavan 3-ol degradation involved the formation of derivatives that retained the properties of their
parent compounds. Since these properties are linked to oxidative- and advanced glycation end-prod-
uct-related diseases, these results suggest that green tea fortification of intermediate moisture apple
products could be a valuable means of product innovation, to address consumers’ nutritional needs.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Most apple production is intended for industrial processing into

Apples are one of the most important fruits in terms of produc-
tion worldwide. The world’s largest apple producer is China, fol-
lowed by the US, then Turkey, Iran, Italy, France, Poland and
Russia (http://www.fao.org/es/ess/top/commodity.html). Apples
are rich in phytochemicals, such as flavan 3-ols (oligomeric procy-
anidins, epicatechin and catechin), hydroxycinnamic acids (chloro-
genic acid and a p-coumaric acid derivative), dihydrochalcones
(phloridzin and phloretin 20 O-xyloglucoside), flavonols (quercetin
glycosides) and anthocyanins (cyanidin glycosides) (Vrhovsek,
Rigo, Tonon, & Mattivi, 2004).
Abbreviations: FRAP, ferric reducing/antioxidant power; DPPH, 2,2-diphenyl-1-
(2,4,6-trinitrophenyl)hydrazyl radical-scavenging activity; AGE, advanced glycation
endproducts; GT, green tea; EGCG, ()-epigallocatechin gallate; EC, ()-epicatechin
(EC); EGC, ()-epigallocatechin; ECG, ()-epicatechin gallate; GCG, ()-gallocate-
chin gallate; GC, ()-gallocatechin; CG, ()-catechin gallate; C, ()-catechin; BSA,
bovine serum albumin; PPO, polyphenol oxidase.
⇑ Corresponding author. Tel.: +39 2 50319172; fax: +39 2 50319191.
E-mail address: vera.lavelli@unimi.it (V. Lavelli).
juice, puree, nectar, cider, vinegar and dehydrated products. Two
types of intermediate moisture (IM) apple products are manufac-
tured. One is a concentrated juice and contains about 25% moisture
(Spanos & Wrolstad, 1992). The second contains 10–25% moisture
and is referred to as evaporated apple. These products are used
either as ingredients for different food products (e.g. for pie filling)
or are remoistened prior to use, or as IM foods (Brennan, 1994).
Processing of apples causes both a decrease in antioxidant con-
tent and browning (Spanos & Wrolstad, 1992). IM apple is stable
from the stand point of fermentation, whereas major deteriorative
quality problems are related to non-enzymatic browning (Burdurlu
& Karadeniz, 2003; Torbido & Lozano, 1984; Vaikousi, Koutsou-
manis, & Biliaderis, 2008) and phenolic oxidation (Lavelli &
Vantaggi, 2009).
This study focused on the fortification of IM apple products with
green tea (GT). The food industry has recently regarded GT as useful
for food fortification, having the intention of improving the health-
promoting properties of food products (Wang, Provan, & Helliwell,
2000). GT phytochemicals are associated with health-promoting ef-
fects. GT leaves are composed of about 10% (w/w) flavan 3-ols, which

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.01.047
590 V. Lavelli et al. / Food Chemistry 127 (2011) 589–595
are referred to as catechins. The most prevalent compounds include
()-epigallocatechin gallate (EGCG), ()-epicatechin (EC), ()-epi-
gallocatechin (EGC), and ()-epicatechin gallate (ECG) (Wang, Pro-
van, & Helliwell, 2003). GT catechins possess very high scavenging
ability against reactive oxygen species. Indeed, various epidemiolog-
ical studies have demonstrated that GT consumption decreases the
occurrence of oxidative-related diseases, such as cancer and cardio-
vascular disease (Thielecke & Boschmann, 2009; Wang & Ho, 2009).
In addition, GT catechins effectively trap carbonyl species, which are
extremely reactive as they can modify lysine, arginine and cysteine
residues of proteins, leading to formation of advanced glycation
end-products (AGEs). Hence, GT catechins have the potential to inhi-
bit the progression of symptoms associated with AGE-related dis-
eases, such as diabetes (Wang, Sun, Cao, & Tian, 2009; Thielecke &
Boschmann, 2009).
The intent of this study was to examine an intermediate mois-
ture apple product that is not currently available to consumers and
may be relevant to the functional foods market. This is a growing
industry, and many food companies have seen a great potential
for products that provide additional physiological benefits beyond
their basic nutrition (Day, Seymour, Pitts, Konczak, & Lundin,
2009). Here, we have studied a new food product made from rela-
tively cheap food ingredients, but that have somewhat valuable
bioactive components in terms of human health protection. Chal-
lenges remain to ensure that functional ingredients remain stable
after food processing and storage (Day et al., 2009).
In fact, during processing and storage of GT drinks, flavan 3-ols
undergo degradation (Labbé, Têtu, Trudel, & Bazinet, 2008). GT fla-
van 3-ols also degrade during long-term storage in the dry state
(Friedman, Levin, Lee, & Kozukue, 2009); therefore a new GT for-
mulation should be evaluated for its stability. Intermediate aw lev-
els are particularly critical for product stability, since moisture
content is sufficient to permit the occurrence of Maillard reactions
and oxidation (Brennan, 1994). The rate of non-enzymatic brown-
ing is high in dehydrated foods, due to elevated concentrations of
reagents, with maximum values in the IM foods with water activity
(aw) levels between 0.5 and 0.8. Below 0.5, the reaction is limited
by low mobility of reactants whereas, above 0.8, the browning rate
decreases with increasing aw due to a dilution effect (Vaikousi
et al., 2008). An increase in aw above 0.3 increases the rate of anti-
oxidant degradation in apples, most likely by increasing mobility of
reactants and bringing catalysts into solution (Lavelli & Vantaggi,
2009). In addition, as the solid matrix swells, new surfaces for oxi-
dation are exposed (Brennan, 1994).
Therefore, the GT flavan 3-ols incorporated into an intermediate
moisture apple matrix are challenged by different reactive car-
bonyl species and free radicals derived from these degradation
reactions.The purpose of this study was to investigate the effect
of GT addition on IM apple quality and stability.
GT-fortified IM apple products were stored in the aw range
0.56–0.75, at 30 °C. Kinetics of colour changes, phytochemical deg-
radation and in vitro antioxidant and anti-glycoxidative properties
were investigated in comparison to unfortified IM apple products.
2. Materials and methods
2.1. Materials
The reagents sodium chloride, 2,2-diphenyl-1-(2,4,6-trinitro-
phenyl)hydrazyl radical (DPPH), 6-hydroxy-2,5,7,8-tetramethylch-
roman-2-carboxylic acid (Trolox), 2,4,6-tripyridyl-s-triazine,
FeSO47H2O, FeCl36H2O, Folin–Ciocalteu phenol reagent, bovine
serum albumin (BSA, 97% fatty acid free), Chelex resin, fructose, so-
dium azide, sodium phosphate monobasic, sodium phosphate
dibasic, and standards of ascorbic acid, caffeine, chlorogenic acid,
phloridzin and gallic acid were purchased from Sigma Aldrich (Mi-
lan, Italy). Standards of C, EC, EGC, EGCG, ECG, GCG, and procyani-
din B2 were purchased from Extrasynthese (Lyon, France).
2.2. Green tea extraction
Java Green Tea (Twinings, London, UK) was purchased at a local
supermarket. In a half-litre Pyrex bottle, GT extract was prepared
by extracting 25 g of dried leaves in 500 ml of deionised water,
which had been pre-heated at 85 °C. The extraction proceeded
for 5 min before the bottle was immersed in ice/water slurry. The
extract was then filtered through Whatman No. 4 filter paper. A
sample of extract was removed and analysed by HPLC for phenolic
content.
2.3. Preparation of GT-fortified and unfortified IM apples
Fresh apples (Malus domestica) of the cultivar Golden Delicious
were purchased from a local supermarket. Each apple was peeled,
cored with seeds removed, and quartered. One half portion was
designated for GT addition and the other for control. Two batches
of apple portions (2 kg each) were placed into a wire mesh basket
and immediately boiled in a deionised water pot at 100 °C for
4 min. After heating, the basket was rapidly removed from the boil-
ing water, cooled by immersion in a 4 °C deionised water bath, and
drained. Apple slices were then blended to a puree consistency in a
K 3000 Braun Multisystem blender (Braun, Kronberg, Germany).
The amount of GT water extract to be added to the apple puree
was chosen in order to obtain (in a single serving of the fortified
apple product) an amount of GT flavan 3-ols equivalent to that
present in a cup of GT infusion. Moisture content of the apple puree
was determined. A 2 kg batch had 200 g of dry solids; therefore it
corresponded to four single serving portions. In fact, a portion size
of 50 g d.w. is consistent with a commercial single-serving of dehy-
drated apple products available in the marketplace. Preliminary
trials were carried out to evaluate the average retention of GT cat-
echins after freeze-drying, which was about 80%. Based on this
information, a 2 kg batch of apple puree was added to 384 ml of
GT extract (which was expected to provide an amount of GT cate-
chins equal to four cups of GT) mixed thoroughly and freeze-dried
to obtain the GT-fortified apple product. Control and GT fortified
apple purees were then spread over stainless steel trays and
freeze-dried in a Lyoflex Edwards freeze drier (Crawley, UK). The
freeze-dried powders were ground in the food processor and
sieved (800 lm pan sieve mesh size).
2.4. Storage study
For the storage study, powders of apple and apple fortified with
GT were weighed into Petri dishes (0.141 g of powder per cm2). The
dishes were then placed onto wire mesh racks that were sus-
pended within airtight plastic boxes above a saturated sodium
chloride solution (aw at 30 °C = 0.751 ± 0.001) or a saturated so-
dium bromide solution (aw at 30 °C = 0.560 ± 0.004). The boxes
were prepared in duplicate for each experimental treatment and
stored at 30 °C over a period of 2 months in a thermostatted heat-
ing cabinet.
2.5. Water activity (aw), moisture content, soluble solids, pH and
titratable acidity
Water activity (aw) of the samples and of the saturated salt solu-
tions was measured in duplicate, every day from the incubation for
5 d, to confirm that the equilibrium aw had been reached. An Aqua-
lab water activity meter was used (Decagon Devices, WA, USA).
 V. Lavelli et al. / Food Chemistry 127 (2011) 589–595
The moisture content of samples, at equilibrium within the
environmental chambers, was determined in duplicate by drying
samples in a vacuum oven at 70 °C and 50 Torr for 18 h (Lavelli,
Zanoni, & Zaniboni, 2007). Results were reported as grams of water
per 100 g of fresh product.
Soluble solids, pH and titratable acidity were determined in
duplicate on freeze-dried samples after dilution with water (0.5 g
of powder in 20 ml). pH was determined using a model 62 pH me-
ter (Radiometer, Copenhagen, Denmark). Soluble solids were
determined at 20 °C, using a model RFM 340 refractometer (Bell-
ingham & Stanley, Ltd., Tunbridge Wells, UK). Results were re-
ported as °Brix (grams of sucrose per 100 g of dry product).
Titratable acidity was determined by titration with 0.1 N NaOH
to pH 8.1. Results were reported as grams of malic acid per 100 g
of dry product (Lavelli & Vantaggi, 2009).
2.6. Colour
Colour evaluation of samples was performed in duplicate. Sam-
pling was performed at 7 regular storage times during incubation.
A model SL-2000 Chromameter (Labo scientifica, Parma, Italy), cal-
ibrated with a white standard was used. The Hunter L⁄, a⁄, and b⁄
coordinates were recorded, which indicated lightness and darkness
(L⁄), redness (+a⁄), greenness (a⁄), yellowness (+b⁄), and blueness
(b⁄).
2.7. Sample extraction
Each sample was extracted in duplicate at four regular storage
times during incubation, using three procedures. An amount of
0.5 g of sample was added to either 10 ml of methanol, 10 ml of
acetone: water (70:30, v/v), or 5 ml of 6% metaphosphoric acid
(containing 1 g l1 of sodium metabisulphite). The extracts were
then vortexed for 2 min and centrifuged (10,000g for 10 min at
15 °C). The supernatant was then removed and filtered through
Whatman No. 4 filter paper. Methanolic extracts were evaluated
for individual phenolic content, total phenolics, antioxidant activ-
ity, and anti-glycation activity. The 70% acetone extracts were eval-
uated for procyanidin content. The 6% metaphosphoric acid
extracts were analysed for ascorbic acid content.
2.8. Phenolic compounds
The determination of phenolic compounds in the methanolic
extracts was conducted by following the methods of Tomas-
Barberan et al. (2001). A model 600 HPLC pump, coupled with a
model 2996 photodiode array detector, operated by Empower soft-
ware (Waters, Vimodrone, Italy), was used. The separation was
performed using a 250  4.6 mm i.d., 5 lm, Symmetry C18 re-
verse-phase column (Waters, Vimodrone, Italy). For preparation
of quaternary mobile phases, 5% formic acid was first added to
stock solutions of methanol and water. Mobile phases were pre-
pared from these solutions as follows: mobile phase A consisted
of water/methanol (95:5, v/v), mobile phase B was water/methanol
(88:12, v/v), mobile phase C was water/methanol (20:80, v/v), and
mobile phase D was 100% methanol. The gradient elution system
was as follows: 0–5 min, 100% A; 5–10 min, linear gradient to
reach 100% B; 10–13 min, 100% B; 13–35 min, linear gradient to
reach 75% B and 25% C; 35–50 min, linear gradient to reach 50%
B and 50% C; 50–52 min linear gradient to reach 100% C; 52–
57 min, 100% C; 57–60 min, 100% D. Injection volume was 20 ll.
All analyses were conducted at 22 °C. The flow rate was
1.0 ml min1.
Standards of chlorogenic acid, EC, procyanidin B2, phloridzin
and caffeine were used to identify peaks and to build calibration
curves for quantification. EC, procyanidin B2, phloridzin and caf-
591
feine were quantified at 280 nm and chlorogenic acid at 330 nm.
Standards of EGCG, EGC, ECG, GCG and C were used to identify
peaks. These flavan 3-ols were quantified by using the calibration
curve built with EC and the relative response factors reported by
Wang et al. (2003). A peak was tentatively assigned to phloretin
20 -O-xyloglucoside based on its UV–vis spectrum and retention
time, and quantified as phloridzin. Concentrations were expressed
as milligrams per kilogram of dry product.
2.9. Ascorbic acid
The ascorbic acid contents of sample extracts in 6% metaphos-
phoric acid, containing 1 g l1 of sodium metabisulphite, were ana-
lysed according to an HPLC procedure described previously (Lavelli
& Vantaggi, 2009). A model 600 HPLC pump, coupled with a model
2996 photodiode array detector, operated by Empower software
(Waters, Vimodrone, Italy) was used. A Bio-Rad Fruit Quality Anal-
ysis column (300  7.8 mm i.d.) was employed. Chromatographic
separation was performed with 1 mM H2SO4 as an eluent under
isocratic conditions and 1 ml min1 flow rate, at room tempera-
ture. Ascorbic acid was quantified at 245 nm, using a calibration
curve built with a pure standard. Concentrations were expressed
as milligrams per kilogram of dry product.
2.10. Procyanidin
The procyanidin contents of apple extracts in acetone:water
(70:30, v/v) were analysed. For sample preparation, an aliquot of
extract (0.25 ml) was dried with nitrogen and re-dissolved into
1 ml of 0.1 M phosphate buffer, pH 7.0. Then, the sample was puri-
fied with a 500 mg Sep-Pak C18 cartridge (Waters, Vimodrone,
Italy) and eluted with 1 ml of methanol (Lavelli & Vantaggi,
2009). To remove chlorophyll interference in the samples contain-
ing GT, 1 ml of hexane was added to 1 ml of the purified extract
and the lower phase was used for the analysis. For the vanillin as-
say, the reaction medium comprised 0.5 ml of sample (after Sep-
Pak-purification) or standard in methanol, 1.25 ml of 1% vanillin
in methanol, and 1.25 ml of 9 N H2SO4 in methanol (Sun, Ricar-
do-da-Silva, & Spranger, 1998). The mixture was incubated at
25 °C until the time of reaching maximum absorbance at 500 nm.
The maximum absorbance at 500 nm was used to quantify procy-
anidin content using a calibration curve built with the monomeric
flavan 3-ol C. A Jasco UVDEC-610 spectrophotometer (Jasco Europe,
Cremella, LC, Italy) was used. Procyanidin content was expressed
as milligrams of C equivalents per kilogram of dry product.
2.11. Folin–Ciocalteu assay
The Folin–Ciocalteu assay was performed on the methanolic ex-
tracts, according to a procedure described previously with slight
modifications (Singleton, Orthofer, & Lamuela-Raventos, 1999).
The reaction mixture contained 6 ml of distilled water, 76 ll of ap-
ple extract or 38 ll of GT-fortified apple extract and 38 ll of ace-
tone:water (70: 30, v/v), 380 ll Folin–Ciocalteu reagent and
1.14 ml of 20% Na2CO3. Samples were incubated for 45 min at room
temperature and then the absorbance was recorded at 760 nm
using a Jasco UVDEC-610 spectrophotometer (Jasco Europe, Crem-
ella, LC, Italy). A calibration curve was built using gallic acid. Total
phenolics were expressed as milligrams of gallic acid equivalents
per kilogram of dry product.
2.12. FRAP assay
The FRAP assay was estimated on the methanolic extracts,
according to a procedure described previously (Benzie & Strain,
1996). Briefly, FRAP reagent was prepared by adding 25 ml of
592 V. Lavelli et al. / Food Chemistry 127 (2011) 589–595
300 mM acetate buffer, pH 3.6, 2.5 ml of 10 mM 2,4,6-tripyridyl-s-
triazine in 40 mM HCl and 2.5 ml of 20 mM FeCl3. The reaction
mixture contained 90 ll of sample and 3 ml of FRAP reagent. The
increase in absorbance at 593 nm was evaluated after 4 min of
incubation at 37 °C, using a Jasco UVDEC-610 spectrophotometer
(Jasco Europe, Cremella, LC, Italy). A methanolic solution of FeSO4
was used for calibration. Results were expressed as millimoles
Fe(II) sulphate equivalents per kilogram of dry product.
2.13. DPPH assay
The DPPH radical-scavenging activity was performed as de-
scribed previously (Lavelli & Vantaggi, 2009). Briefly, 1 ml of differ-
ent dilutions of the sample methanolic extracts was added to
2.5 ml of a 6.35  105 M methanolic solution of DPPH. The de-
crease in absorbance was determined at 515 nm after a 30 min
incubation at room temperature (when a constant value was
reached), using a Jasco UVDEC-610 spectrophotometer (Jasco
Europe, Cremella, LC, Italy). The percent decrease of DPPH concen-
tration was calculated with respect to the initial value. A dose–
response curve was constructed, and the amount of sample
required to lower the initial DPPH concentration by 50%, I50, was
interpolated. Trolox was used as a reference antioxidant. The
DPPH-scavenging activity of apple powders was expressed as Trol-
ox equivalents (TE). TE is the ratio of the I50 of Trolox (nanomoles)
to the I50 of the sample (milligrams, dry weight).
2.14. Polyphenoloxidase activity (PPO)
PPO was determined on freeze-dried products, following the
methods by Alvarez-Parrilla et al. (2007). Extracts were first pre-
pared in duplicate by adding 0.10 g of sample to 1 ml of a 0.03 M
acetic acid, 0.14 M K2HPO4, pH 6.5 buffer containing 1 M NaCl,
and 5% (w/w) polyvinylpolypyrrolidone. The mixture was centri-
fuged at 10,000g for 10 min at 20 °C. The supernatant was filtered
through Whatman No. 1 filter paper. For PPO activity evaluation,
0.1 ml of the supernatant was added to 0.9 ml of 10 mM chlorogen-
ic acid in the above-indicated buffer. Unblanched apple extract was
used as a control. The reaction mixture was incubated at 25 °C, and
the rate was estimated from the linear increase in absorbance at
400 nm, using a Jasco UVDEC-610 spectrophotometer (Jasco Eur-
ope, Cremella, LC, Italy).
2.15. Fructose-induced glycation of BSA assay
The inhibition of glycation of BSA was conducted with the
methanolic extracts according to the procedure described by
McPherson, Shilton, and Walton (1988) with modification. The
reaction mixture consisted of 50 ll of sample or standard, 900 ll
of phosphate buffer (200 mM potassium phosphate buffer, pH
7.4, with 0.02% sodium azide, treated with Chelex resin), 300 ll
of BSA solution (50 mg ml1 of BSA in Chelex-treated phosphate
buffer), and 300 ll of fructose solution (1.25 M fructose in che-
lex-treated phosphate buffer). A control reaction was also incu-
bated with no addition of sample or standard. Tubes were
capped and incubated at 38 °C for 72 h. Following incubation, sam-
ples were analysed for fluorescence on a Perkin-Elmer LS 55 Lumi-
nescence Spectrometer (Perkin-Elmer, United Kingdom) with an
excitation/emission wavelength pair k = 370/440 nm with 5 nm slit
width, read against incubated phosphate buffer blank. Each stan-
dard or sample was evaluated at five dilutions, in triplicate over
two separate days. The monomeric flavan 3-ol C dissolved in meth-
anol (final concentrations in the assay mixture: 10–90 nmol ml1)
was evaluated as an external standard. Results were reported as %
inhibition of fructose-induced glycation of BSA, where % inhibi-
tion = 100  (fluorescence intensity of sample or standard  inten-
sity of BSA)/(intensity of control reaction  intensity of BSA). Data
points of concentration versus % inhibition were plotted and an
estimate of inhibition of protein glycation by 30% (IC30) was se-
lected, since this level of inhibition occurred within the linear
range of all samples evaluated. Results were reported as catechin
equivalents, CE, which was the ratio of I30 of catechin (nanomoles)
to I30 of sample (milligrams, dry weight).
2.16. Statistical analysis
Results represent the average values of duplicate determina-
tions made on independent aliquots for each sample. Data regres-
sion and analysis of variance were conducted using the
Statgraphics 5.1 software (STCC Inc., Rockville, MD); Fisher’s least
significant difference (LSD) procedure (p < 0.05) was used to dis-
criminate among the means.
3. Results and discussion
3.1. Moisture content, titratable acidity, pH, soluble solids content and
colour of IM apple and GT-fortified apple
When the unfortified and GT-fortified apple products were
stored at constant environmental humidity values of 56% and
75%, their aw levels approached those of the saturated salt solu-
tions after 3 d of incubation, and within 5 d of incubation the equi-
librium aw levels were reached. GT addition did not affect the
equilibrium moisture contents of apple, which were 15% and 26%
at the aw levels of 0.56 and 0.75, respectively (Table 1). This result
indicated that GT addition did not change apple hygroscopicity,
probably because the GT:apple ratio was low (about 6 mg of GT
catechins per g of dry apple). These apple products therefore
matched IM apples (Brennan, 1994).
The titratable acidity, pH, and soluble solids content were eval-
uated in diluted freeze-dried apple and GT-fortified apple. On a
d.w. basis, the titratable acidity was 2.52 ± 0.03 g malic acid/
100 g of product in the unfortified apple and increased in GT-forti-
fied apple to 2.76 ± 0.01 g malic acid/100 g of product. These values
were significantly different (p < 0.05), although this variation can
be considered minimal. pH was not affected by GT addition
(p > 0.05); the average pH of the unfortified and GT-fortified apple
was 3.65 ± 0.09. Soluble solids content was also not significantly
different (p > 0.05) between the GT-fortified apple and unfortified
apple, with an average value of 75.1 ± 2.9 g sucrose/100 g d.w.
It is worth noting that apples contain organic acids which may
affect GT catechin stability; however, prediction of the interplay
among pH, organic acids and GT catechins is not feasible. Previous
studies have indicated that, in general, long-term stability of GT
catechins decreases when pH is increased (Chen, Zhu, Tsang, &
Huang, 2001). Nevertheless, in citrate buffer, catechin stability in-
creases with increasing pH from 2.6 to 5.6 (Tu & Watanabe, 2005).
Citric acid addition to a powdered GT formulation resulted in sig-
nificant degradation of total catechins at P58% relative humidity,
due to the pH of 1.6 of the formulation (Ortiz, Ferruzzi, Taylor, &
Mauer, 2008), which is much lower than that of IM apples.
Table 1
Equilibrium moisture content of IM apples.
aw level Equilibrium moisture content (%)
Apple GT-Apple
0.56 15.0 ± 0.4 14.7 ± 0.2
0.75 26.3 ± 0.12 26.3 ± 0.2
Results are averages ± SD.
 V. Lavelli et al. / Food Chemistry 127 (2011) 589–595 593

The colour of apple products was analysed. The values of the L⁄
(0 = blackness, 100 = whiteness), a⁄ (a⁄ = greenness, +a⁄ = red-
ness), and b⁄ (b = blueness, +b = yellowness) colourimetric
parameters are reported after 5 d of storage, when the equilibrium
aw levels of 0.56 and 0.75 were reached (Table 2). At this time, the
L⁄ and b⁄ values for GT-fortified IM apple were slightly lower than
those of the IM apple, while the a⁄ value was slightly higher. These
trends indicated that GT addition slightly darkened the apple col-
our. Changes in the colourimetric parameters were fitted to pseu-
do-zero-order kinetic models, with changes occurring over time
indicated by variation in the rate constant k (Table 2). During stor-
age at constant aw levels, both the GT-fortified IM apple and the IM
apple became increasingly browner, with a higher rate for colour
variation at the aw level of 0.75 than at 0.56. The L⁄ value decreased
with the same rate in GT-fortified and unfortified apple, whereas
the rates of variation of the a⁄ and b⁄ values were faster in the
IM apple product than in the GT-fortified apple. These results indi-
cate that the mechanisms for browning were different between
GT-fortified and unfortified IM apples. Since products were
blanched and no residual PPO activity was detectable in the sam-
ples (data not shown), changes in colour could be attributed to
non-enzymatic browning reactions. The occurrence of the Maillard
reaction in IM apples has been demonstrated (Burdurlu & Karade-
niz, 2003; Torbido & Lozano, 1984; Vaikousi et al., 2008). This reac-
tion also involves ascorbic acid and phenolic degradation (Nicoli,
Anese, & Parpinel, 1999). EC and EGCG have been demonstrated
to reduce the extent of the Maillard reaction in a glucose–glycine
model system (Noda & Peterson, 2007). Similar inhibition could
have occurred in GT-fortified apple product. It is worth noting,
however, that in the mentioned glucose–glycine model system,
catechins were added in equimolar concentration while, in our
condition, GT catechins were present in very small amounts with
respect to sugars and proteins. In addition, as tea is subjected to
processing and storage, it may form brown-coloured compounds
that are products of oxidation and polymerisation of catechins
(Manzocco, Anese, & Nicoli, 1998). Interestingly, the resultant ef-
fect of these reactions was that when GT was incorporated into ap-
ple, the rates of a⁄ and b⁄ variation diminished slightly and the rate
of L⁄ decrease was not affected.
3.2. Phenolic compounds of IM apple and GT-fortified apple
The IM GT-fortified apple product was intended to provide, per
single serving (50 g d.w.), an amount of flavan 3-ols equivalent to
that present in a cup of GT. EGCG content is a marker of GT quality,
since it is often present as the predominant flavan 3-ol compound
among various GT varieties and has significant bioactive properties
(Wang, Zhou, & Jiang, 2008). As expected, EGCG content of a single
serving of apple products, equilibrated at the aw levels of 0.56 and
0.75, was 115 mg (Table 3), which is consistent with the amount
found in a cup of GT (which is typically 55–110 mg) (Reto, Figueira,
Filipe, & Almeida, 2007).
The degradation of phytochemicals of GT-fortified and unforti-
fied IM apple products was modelled following a pseudo-first-or-

Table 2
Colourimetric parameters in IM unfortified and GT-fortified apples stored at aw levels of 0.56 and 0.75 at 30 °C, in the dark for 5 d and rate constants for colour variation during
storage over two months.

Colourimetric parameter Sample aw 0.56 aw 0.75

1
C5d (CU) k (CU d ) R C5d k (CU d1) R
⁄
L Apple 75.0 ± 0.8 0.139 ± 0.015 0.89 73.2 ± 2 0.180 ± 0.033 0.76
GT-Apple 71.5 ± 0.8 0.120 ± 0.015 0.84 69.2 ± 2 0.179 ± 0.021 0.85
a⁄ Apple 0.94 ± 0.19 0.088 ± 0.003 0.98 0.66 ± 0.25 0.140 ± 0.004 0.99
GT-Apple 0.08 ± 0.02 0.069 ± 0.003 0.97 0.25 ± 0.27 0.121 ± 0.005 0.97
b⁄ Apple 26.1 ± 0.7 0.133 ± 0.012 0.91 25.1 ± 1.2 0.147 ± 0.025 0.78
GT-Apple 23.5 ± 0.4 0.078 ± 0.006 0.93 22.9 ± 0.5 0.105 ± 0.010 0.89

Data were fitted to pseudo-zero-order kinetics: C = C0 + kt; p < 0.01. CU, colourimetric units; k, rate constant; R, correlation coefficient. Data are mean values ± standard error.

Table 3
Antioxidant contents in IM unfortified and GT-fortified apples stored at aw levels of 0.56 and 0.75 at 30 °C, in the dark for 5 d, rate constants for antioxidant degradation during
storage over two months and predicted half-life values.

Compound Sample aw 0.56 aw 0.75

C5d (mg/kg d.w.) k (d1) R t1/2 (d) C5d (mg/kg d.w.) k (d1) R t1/2 (d)
EGC GT-Apple 1900 ± 100 0.010 ± 0.001 0.93 69 1900 ± 300 0.011 ± 0.001 0.96 63
EGCG GT-Apple 2300 ± 300 0.013 ± 0.002 0.96 53 2270 ± 320 0.020 ± 0.001 0.99 35
ECG GT-Apple 440 ± 60 0.014 ± 0.002 0.88 50 470 ± 90 0.013 ± 0.004 0.71 53
GCG GT-Apple 520 ± 20 0.003 ± 0.001 0.61 231 510 ± 60 0.009 ± 0.001 0.96 77
EC Apple 260 ± 30 0.021 ± 0.002 0.96 33 240 ± 30 0.034 ± 0.002 0.99 20
GT-Apple 780 ± 50 0.014 ± 0.001 0.96 50 760 ± 60 0.014 ± 0.001 0.95 50
C Apple 44 ± 5 0.045 ± 0.004 0.98 15 38 ± 11 0.072 ± 0.012 0.96 10
GT-Apple 300 ± 15 n.s.   300 ± 23 n.s.  –
Procyanidin B2 Apple 390 ± 50 0.038 ± 0.002 0.99 18 322 ± 70 0.055 ± 0.005 0.97 13
GT-Apple 460 ± 40 0.015 ± 0.002 0.96 46 396 ± 70 0.025 ± 0.007 0.82 28
Tot. Procyanidins Apple 1470 ± 450 0.014 ± 0.005 0.82 50 1400 ± 200 0.013 ± 0.003 0.92 53
GT-Apple 2250 ± 20 0.013 ± 0.001 0.99 53 2300 ± 200 0.011 ± 0.001 0.99 63
Phloretin glyc. Apple 130 ± 10 0.003 ± 0.001 0.91 231 130 ± 20 0.005 ± 0.001 0.88 139
GT-Apple 130 ± 10 0.011 ± 0.003 0.83 63 130 ± 10 0.010 ± 0.003 0.86 69
Chlorogenic acid Apple 1050 ± 20 0.002 ± 0.001 0.97 347 1130 ± 70 0.005 ± 0.001 0.86 139
GT-Apple 1070 ± 30 0.004 ± 0.001 0.85 173 1050 ± 50 0.007 ± 0.001 0.94 99
Ascorbic acid Apple 89 ± 3 0.058 ± 0.001 0.099 12 11 ± 2 0.481 ± 0.038 0.99 1. 5
GT-Apple 100 ± 10 0.049 ± 0.003 0.99 14 10 ± 2 0.501 ± 0.025 0.99 1.3

n.s. = No significant degradation occurred during storage. Tot. procyanidins were determined by the vanillin assay. Phloretin glyc. = sum of phloridzin + phloretin 2’O-
xyloglucoside. Data were fitted to pseudo-first-order kinetics: ln C = ln C0 + kt; p < 0.01. k, rate constant: t1/2, predicted half life time; R, correlation coefficient. Data are mean
values ± standard error.
594 V. Lavelli et al. / Food Chemistry 127 (2011) 589–595
der kinetic model. The degradation rate constants k were calcu-
lated, along with the correlation coefficients (R), providing an indi-
cation of the model fit for the degradation of each analyte (Table 3).
In the GT-fortified product at aw 0.56, the epi-isomers of cate-
chins degraded with similar rates, with half-life values in the range
of 50–63 d, whereas C and GCG were more stable. EGCG and GCG
degradation rates accelerated with increasing aw to 0.75, and
half-life values became 35 and 77 d, respectively.
In water solution, two major reactions are involved in EGCG
degradation: one is epimerisation, which occurs at the C2 position
and generates GCG; the other is auto-oxidation, involving the B-
ring and leading to the formation of EGCG dimers, whose structure
was elucidated (Sang, Lee, Hou, Ho, & Yang, 2005). Auto-oxidation
is prevailing unless inhibition is achieved with nitrogen conditions
or superoxide dismutase (Sang et al., 2005). Beside formation of
homo-dimers, EGCG can also form hetero-dimers with both EGC
and ECG (Nielson et al., 2007). It was suggested that catechins con-
taining either pyrogallol (e.g. EGCG and EGC) or catechol (e.g. ECG)
B-ring structures are adequate to allow dimerisation; however, the
former compounds react faster. Dimerisation of catechins is signif-
icantly slower when the pH is decreased from 7 to 2 (Nielson et al.,
2007).
In this study, the GT-apple product was exposed to oxygen; in-
deed the content of GCG decreased, indicating that epimerisation
was not the prevailing mechanism for EGCG degradation. It could
be postulated that auto-oxidation occurred in the GT-fortified IM
apple product, with mechanisms similar to those observed in
water solutions. While the pH of the apple product could have de-
creased catechin degradation rates, elevated reactant concentra-
tions in intermediate moisture conditions would support
degradation.
The degradation rates of the monomeric compounds C and E
and of the dimeric procyanidin B2 were significantly higher in IM
apple that in the GT-fortified IM apple. Likely, these compounds
in the GT-fortified product, competed with GT-catechins, since
they can participate in the same degradation reactions. In fact, at
aw 0.56 the half-life values of these compounds were 15, 33 and
18 d for apple and not detectable, 50 and 46 for GT-fortified apple.
At aw 0.75, the degradation rates for C and E and procyanidin B2
were higher in both the fortified and unfortified apples than at
an aw of 0.75. The total procyanidins (determined by the vanillin
assay) degraded at similar rates in the GT-fortified and unfortified
apple products.
The rates for dihydrochalcones and chlorogenic acid degrada-
tion were higher in the GT-fortified apple than in apple products,
but relatively lower compared to those of catechins. It could be
hypothesised that these compounds interacted with GT catechins
by a coupled oxidation mechanism. Ascorbic acid had very poor
stability, in both the GT-fortified and in the unfortified IM apple
Table 4
Anti-AGE formation activity, total phenolics, FRAP values and DPPH-scavenging activity of IM unfortified and GT-fortified apples stored at aw
level of 0.75 at 30 °C, in the dark for 5 d, rate constants for their variations during storage over two months and predicted half-life values.
Assay Sample
Anti-AGE Apple
(mmol CE/kg d.w.) GT-Apple
Total phenolics Apple
(mg GAE/kg d.w.) GT-Apple
FRAP Apple
(mmol FE II/kg d.w.) GT-Apple
DPPH Apple
(mmol TE/kg d.w.) GT-Apple
n.s. = No significant degradation occurred during storage. Data were fitted to pseudo-first-order kinetics: ln C = ln C0 + kt; p < 0.01. k, rate
constant; t1/2, predicted half life time; R, correlation coefficient. Data are mean values ± standard error.
products, and decreased with similar rates having half-life values
of about 13 and 1.5 at aw values of 0.56 and 0.75, respectively.
Therefore, even though it has been reported to stabilise catechin
in aqueous solution (Chen, Zhu, Wong, Zhang, & Chung, 1998) at
intermediate aw levels, its contribution to long-term catechin sta-
bility is likely unimportant.
The caffeine content in GT-fortified apple was 1273 ± 100 mg/
kg d.w., and remained constant through storage.
3.3. In vitro antioxidant activity and inhibition of fructose-induced
glycation of BSA
Total phenolics, ferric reducing antioxidant potential values and
DPPH radical-scavenging activity were about 3–5-times greater for
the GT fortified product than for the control, when the equilibrium
aw was reached (Table 4). In the flavan 3-ol structure, the ortho-tri-
hydroxyl group in the B ring and the galloyl moiety at the C3 posi-
tion of the C ring (e.g. EGCG, GCG, ECG) are the most important
structural features for displaying an excellent scavenging ability
on either DPPH or other radicals (Nanjo et al., 1996).
The decrease of these properties in the GT-fortified and unforti-
fied products was studied by applying a pseudo-first order kinetic
model. After storage at aw of 0.75, the decrease was significant
(p < 0.01), in both the GT-fortified and unfortified IM apple prod-
ucts (Table 4). Whatever the measuring system, these properties
decreased to a lesser extent in the GT-fortified apple product (half
time values were in the range 165–430 d) than in the apple prod-
uct (half time values were in the range 80–130 d). It is worth not-
ing that the Folin–Ciocalteu reducing capacity, FRAP values, and
DPPH radical-scavenging activity of the unfortified and GT-fortified
IM apple products showed only moderate decreases during storage
if compared with the decrease in the antioxidant compounds.
The inhibitory activity against protein glycation was about 3-
fold greater in the GT-fortified apple product than in the control
IM apple at the time of equilibration. The pattern of protein glyca-
tion is complex, starting from reactions between reducing sugars
and amino groups of protein, followed by structural rearrange-
ments to form AGEs. Some phenolic extracts can inhibit these reac-
tions (Rudnicki et al., 2007; Ho, Wu, Lin, & Tang, 2010; Tsuji-Naito,
Saeki, & Hamano, 2009; Wang et al., 2009). In an aqueous glucose/
glycine model system EC, ECG and EGCG were reported to inhibit
glycation by acting through carbonyl trapping, which involves
the A-ring (the meta-polyhydroxylated benzene ring) (Noda & Pet-
erson, 2007). In a protein-glucose model system, Nakagawa,
Yokozawa, Terasawa, Shu, and Juneja (2002) found that GT cate-
chins containing a gallate moiety at the C-3 position of the C-ring
(e.g. EGCG, GCG, and ECG) have significantly greater efficacy in
inhibiting glycation, whereas the presence of an ortho-trihydroxyl
aw 0.75
C5d (mg/kg d.w.) k (d1) R t1/2 (d)
15 ± 2 0.0085 ± 0.001 0.70 81
48 ± 3 n.s. – –
6600 ± 500 0.0082 ± 0.001 0.90 84
15400 ± 700 0.0023 ± 0.0008 0.71 301
71 ± 3 0.0053 ± 0.001 0.54 130
240 ± 10 0.0016 ± 0.001 0.50 430
13 ± 1 0.0084 ± 0.001 0.97 83
64 ± 5 0.0042 ± 0.001 0.84 165
 V. Lavelli et al. / Food Chemistry 127 (2011) 589–595
or an ortho-dihydroxyl in the B ring is not a distinctive structural
feature for this property.
In this study, the anti-glycation activity did not decrease signif-
icantly during storage in the GT-fortified apple, whereas it did de-
crease in the control IM apple. To our knowledge, this is the first
report in which significant anti-glycation activity was found in a
GT product after storage under unfavourable environmental condi-
tions, which caused significant decrease in monomeric flavan 3-ol
contents. This result suggests that catechin degradation in the
GT-fortified product involved the formation of derivatives that re-
tained the properties of their parent compounds. It is also consis-
tent with the hypothesis that catechins dimerised through
condensation of the B-ring of the molecule, as discussed in the pre-
vious paragraph. Investigation of the patterns of GT catechin deg-
radation, and on the biological effects of their derivatives,
deserves further consideration. It is worth noting, however, that
both GT and black tea, which contains simple and oxidised cate-
chins, decrease the level of glycated lysine in the plasma of diabetic
rats, though only GT also causes a significant decline in plasma tri-
glycerides (Vinson & Zhang, 2005).
4. Conclusions
In unfortified and GT-fortified IM apples (about 6 mg of GT cat-
echins added per g of dry apple), water availability (aw levels of
0.56 and 0.75) was sufficient to support various chemical reactions
during storage at 30 °C. Colour variation occurred with slightly
lower rates after GT addition. Antioxidant and anti-glyco-oxidative
properties decreased in IM apple. After addition of GT extract, anti-
oxidant and anti-glycoxidative properties rose by 3- and 5-fold,
respectively. Incorporated GT catechins underwent degradation
during storage; however, the antioxidant activity decreased at a
slow rate and the anti-glycoxidative properties were retained over
two months. Since these properties are linked to oxidative- and
AGE-related diseases, these results suggest that GT fortification
of IM apple could be a valuable means of product innovation to ad-
dress consumers’ nutritional needs.
Acknowledgement
This study was funded in part by USDA grant # 2005-38420-
15693.
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