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Investigation of the antioxidant capacity of caramels: Combination of laboratory assays and C. elegans model

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Caramel is one of the most current additive/ingredient obtained by controlled heat treatment of carbohydrates. A combination of in vitro and in vivo methods showed that aromatic caramels and caramel colours are antioxidant. The highest value is obtained for Class III caramel colours for in vitro TEAC and KRL tests (401 µmol Trolox equivalent/g and 5 366 mg gallic acid equivalent/100 g, respectively). In in vivo test, caramels also increase the resistance of C. elegans to oxidant attack, with until 40% live worms after 18 h of treatment in the presence of caramels against no live worms after 12 h of treatment without. In conclusion, this study explore benefit effect of caramels and showed an antioxidant activity linked to their color intensity. The molecules involved remain to be explored.
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Journal of Functional Foods 78 (2021) 104308
Available online 25 January 2021
1756-4646/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Investigation of the antioxidant capacity of caramels: Combination of
laboratory assays and C. elegans model
C´
edric Moretton
a
,
*
, C´
ecile Gouttefangeas
a
, Constance Dubois
b
, Fr´
ed´
eric Jacques Tessier
b
,
Chantal Fradin
b
, Emmanuelle Prost-Camus
c
, Michel Prost
c
, Marc Haumont
c
, Henri Nigay
a
a
NIGAY SAS, F-42110 Feurs, France
b
Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167 - RID-AGE - Facteurs de risque et d´
eterminants mol´
eculaires des maladies li´
ees au vieillissement, F-59000
Lille, France
c
LARA-SPIRAL, F-21560 Couternon, France
ARTICLE INFO
Keywords:
Caramel
Antioxidant
TEAC
KRL
C. elegans
ABSTRACT
Caramel is one of the most current additive/ingredient obtained by controlled heat treatment of carbohydrates.
A combination of in vitro and in vivo methods showed that aromatic caramels and caramel colours are anti-
oxidant. The highest value is obtained for Class III caramel colours for in vitro TEAC and KRL tests (401 µmol
Trolox equivalent/g and 5 366 mg gallic acid equivalent/100 g, respectively). In in vivo test, caramels also in-
crease the resistance of C. elegans to oxidant attack, with until 40% live worms after 18 h of treatment in the
presence of caramels against no live worms after 12 h of treatment without.
In conclusion, this study explore benet effect of caramels and showed an antioxidant activity linked to their
color intensity. The molecules involved remain to be explored.
1. Introduction
The word caramelrefers to various products depending on recipes
and applications (confectionaries, ingredients for avouring, additives
for colouring). Aromatic caramels and caramel colours are widely used
in the food industry for their technological properties. Aromatic cara-
mels are light to dark brown liquids or solids considered as food in-
gredients. Their main functionality is to enhance taste in diverse food
applications including dairy products, ice creams, sauces or spirits.
Caramel colours are used to provide a yellow to brown color in a large
range of foodstuffs. These latter have been classied into 4 classes of
food additives: Class I-IV (or E150a, b, c and d at the European Union
level) according to the manufacturing reactants used. Caramels are
produced by heating carbohydrates from vegetable sources (glucose,
sucrose, invert sugar, etc.) in the presence or not of caramelization
promoters (acids, alkalis, ammonium or sulte compounds). The car-
amelization process includes a multitude of chemical reactions leading
to the formation of a complex mixture of molecules, with low molecular
weight ones responsible for the aromatic feature and high molecular
weight ones (melanoidins) responsible for the colourant feature of car-
amels (Golon & Kuhnert, 2012; Licht et al., 1992; Moretton, 2009;
Sengar & Sharma, 2014). These reactions are specic in heat treatments
and nature of the used sugar.
On the one hand, caramel colours are subject to specic monitoring
due to the presence of adverse constituents at levels that may be of safety
concern in a context of high-level exposure: 2-acetyl-4-tetrahydroxybu-
tylimidazole (THI) and 4-methylimidazole (4-MEI). These molecules are
regulated by national and international authorities at levels of the ppm
range. But on the other hand, some molecules of caramel have been
associated with nutritional and health benets. Studies have shown
potential prebiotic effects for fructose dianhydrides and some other ol-
igosaccharides, present in the order of several hundred grams/kg in
some caramels (Arribas et al., 2010; Orban, Patterson, Sutton, &
Richards, 1997; Peinado et al., 2013; Rubio et al., 2014; Saito & Tomita,
2000). Caramels have also been recently highlighted as potential anti-
oxidants (Sengar & Sharma, 2014). Antioxidants are molecules that
counteract the harmful effects of free radicals and other oxidants,
responsible for causing a large number of diseases by oxidizing nucleic
Abbreviations: ABTS, 2, 2
-azinobis3-ethylbenzothiazoline-6-sulfonic acid; AC, Aromatic caramel; BS, Burnt Sugar; CC, Caramel Colour; EBC colour, European
Brewing Convention colour; GAEq, Gallic Acid Equivalent; KRL, Kit Radicaux Libres; TEAC, Trolox Equivalent Antioxidant Capacity.
* Corresponding author.
E-mail address: cedric.moretton@nigay.com (C. Moretton).
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff
https://doi.org/10.1016/j.jff.2020.104308
Received 12 August 2020; Received in revised form 9 November 2020; Accepted 21 November 2020
Journal of Functional Foods 78 (2021) 104308
2
acids, proteins, lipids and DNA. Antioxidants inhibit or delay the
oxidative process by blocking both the initiation and propagation of
oxidizing chain reactions. There are endogenous antioxidants (super-
oxide dismutase, glutathione peroxidase ) and antioxidants from
exogenous food sources (vitamins C and E, carotenoids, β-carotene,
polyphenols ) (Finley, Kong, Hintze, & Je, 2011).
Brenna et al. aimed to ascertain the contribution to the antioxidant
activity of some caramel-containing soft drinks, such as cola drinks, and
chinotto, an original Italian soft drink (Brenna, Ceppi, & Giovanelli,
2009). The results showed that even if soft drinks have a lower antiox-
idant activity than other beverages such as tea, coffee or chocolate, they
may contribute to the antioxidant pool assumed with the diet. Tsai et al.
heated 4 sugars (including monosaccharides and disaccharides) with
different concentrations (140%) at pH 3, 7, and 10 at 90 C for various
durations (042 h). Results from 240 samples indicated that alkaline
condition (pH 10 vs pH 3) as well as high sugar concentration (40% vs
20%) preferably enhance the antioxidant capacity of caramels, evalu-
ated by ferric reducing antioxidant power (FRAP) method and DPPH
(2,2-diphenl-1-picrylhydrazyl) scavenging ability (Tsai, Yu, Chen, Liu, &
Sun, 2009). A correlation between browning intensity and antioxidant
was also shown.
Compared to the caramels previously studied under experimental
conditions, caramels manufactured by industrial processes are heated at
higher sugar concentration and to higher temperatures but for shorter
durations. In this context, the objective of this study was to evaluate the
global antioxidant capacity of different types of caramels marketed as
aromatic ingredients or food additives. To demonstrate the global
antioxidant capacity of caramels, we used in vitro methods well vali-
dated; the TEAC/ABTS assay (Trolox equivalent antioxidant capacity /
2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) and the KRL(Kit
Radicaux Libres) tests, respectively. The nematode Caenorhabditis ele-
gans was used as an in vivo model for studying caramel-dependent
resistance to oxidative stress.
2. Material and methods
2.1. Materials
2.1.1. Samples
Aromatic caramels and caramel colours were provided by Nigay SAS
(Feurs, France).
Table 1 shows composition and European Brewing Convention (EBC)
colour of the caramel samples. Different raw materials were tested: su-
crose for aromatic caramel and burnt sugars, sucrose or glucose for
caramel colours with an addition of reactants such as alkali, sulte or
ammonia.
The colour intensity and the colouring power increase depending on
the type of caramel and the raw materials used (EBC range from 570
(aromatic caramel) to 46 000 (class IV caramel colour)).
2.1.2. Chemicals
ABTS, potassium persulfate, ascorbic acid, di-potassium hydro-
genophosphate and potassium dihydrogenophosphate were purchased
from VWR chemicals (Fontenay sous bois, France). 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox), Paraquat dichloride
hydrate (PQ) and trivalent Arsenic chloride (AsIII) were purchased from
Acros Organics (Geel, Belgium). Gallic acid and tert-Butyl hydroperoxide
70% aqueous solution (TBHP) were purchased from Alfa Aesar (Kandel,
Germany), KRLtest kit from Kirial International (Couternon, France).
2.1.3. C. elegans and bacterial strains
The Bristol N2 wild type strain and the OP50 Escherichia coli feeding
strain were obtained from the Caenorhabditis Genetics Center (cgc.umn.
edu).
2.2. Methods
2.2.1. Colour intensity EBC units
The EBC colour, initially used for beer, is a method developed by the
Institute of Brewing and the European Brewing Convention to measure
the colour of products. The caramel is diluted with a factor of dilution to
obtain a colour visually comparable with a colour scale (colored stan-
dard discs from 6 to 27, from light yellow to dark brown). The EBC value
is then calculated by the following formula:
EBC =disc value *25/(solution concentration *optical pathlength)
2.2.2. Antioxidant capacity determinations
2.2.2.1. Trolox equivalent antioxidant capacity (TEAC/ABTS assay).
Various chemical methods are used for the evaluation of the antioxidant
capacity of foods (Alam, Bristi, & Raquzzaman, 2013; Pisoschi &
Negulescu, 2012). Most of them use colorimetric technique. The
absorbance of caramels in UV and visible region decrease as wavelength
increases so the choice fell on the TEAC/ABTS method with a color
measurement at high wavelength (734 nm).
The determination of ABTS radical inhibition was based on the
method of Re et al. (Re et al., 1999). Stock water solutions of ABTS and
potassium persulfate were prepared at 5 mmol/L and 2 mmol/L,
respectively. The radical cation ABTS
.+
was obtained by reacting ABTS
solution with potassium persulfate solution (50/50). Before use, the mix
was kept at room temperature in darkness for 1216 h.
One hundred and fty microliters of ABTS
.+
solution was diluted in
4.85 mL phosphate buffer 100 mM pH7 to obtain a blank absorbance
around 0.8 at 734 nm. Absorbance was read using Thermo AquaMate
spectrophotometer.
Antioxidants reduce the blue ABTS radical cation to its colorless
neutral form. Trolox, a water-soluble analog of vitamin E, was used as a
standard antioxidant to draw a linear correlation between the absor-
bance decrease and the concentration of Trolox in µmol. One hundred
microliters of the sample solution were added to the ABTS
.+
/phosphate
buffer mix. The sample is diluted appropriately to produce between 20%
and 80% inhibition of the blank absorbance.
Results were expressed as µmol Trolox equivalents /g of sample
(TEAC).
2.2.2.2. Kit Radicaux Libres (KRL) test. The antioxidant activity of
caramel solutions was tested using the patented KRL test (Phan-Thi,
Durand, Prost, Prost, & Wach´
e, 2016; Prost, 1992). The biological KRL
test is based on the ability of blood cells to resist against free radicals.
Initially used to allow the dynamic evaluation of the overall antioxidant
defense potential of an individual, it is also used for the assessment of
antioxidant capacity of plant extracts and many other antioxidants
products (Brahmi et al., 2018; Cases et al., 2017; Phan-Thi et al., 2016).
Table 1
Raw materials and EBC colour of caramel samples.
Caramel Abbreviation Raw materials EBC
colour
Aromatic caramel AC Sucrose 570
Burnt sugar BS Sucrose 11,000
Class I caramel
colour
CC-I Sucrose - alkali 17,000
Class II caramel
colour
CC-II Sucrose - sodium sulte 19,000
Class III caramel
colour
CC-III a Glucose - ammonium
hydroxide
22,000
Class III caramel
colour
CC-III b Glucose - ammonium
hydroxide
35,000
Class IV caramel
colour
CC-IV a Glucose - ammonium sulte 21,000
Class IV caramel
colour
CC-IV b Glucose - ammonium sulte 46,000
C. Moretton et al.
Journal of Functional Foods 78 (2021) 104308
3
A control blood was subjected to free radical attack under controlled
and standardized conditions with the KRL Reader (37 C, orbital
shaking) in the presence of caramel.
A caramel solution (120 µL, 2250 mg/L) was mixed with whole horse
blood solution (50 µL) and KRL Reagent solution (100 µL) in 96-wells
plate. Gallic acid was used as a reference compound in antioxidant ac-
tivity assessment.
Measuring the decrease in absorbance (620 nm) allows the moni-
toring of the progressive disappearance of the cells. The resistance of
blood cells against free radical attack is expressed by the time necessary
to lyse 50% of the blood cells (HT50 in min).
Results of antioxidant activity (KRL AO) were expressed in mg Gallic
Acid Equivalent /100 g of caramel (GAEq mg/100 g).
2.2.2.3. Caenorhabditis elegans model
2.2.2.3.1. C. elegans culture. Worms were maintained at 20 C on
nematode growth medium (NGM) agar plates seeded with OP50 bac-
teria. Before each experiment, young adult worms were obtained after
synchronization of eggs released from gravid worms by lysis with so-
dium hypochlorite. Except for maintenance and larval development,
feeding bacteria were 10 times concentrated and heat-killed 30 min at
65 C.
2.2.2.3.2. Survival assays in severe oxidative stress. Three days adults
were incubated 24 h at 20 C in 24-well plates (approximately 30 worms
/well) plus NGM containing none, 0.04%, 0.2% and 1% caramel solution
with FUdR (50
μ
M) (FUdR was used to inhibit the development of
progeny) and heat-killed bacteria. Three different oxidizing chemicals,
Paraquat (PQ), tert-Butyl hydroperoxide (TBHP) or trivalent Arsenic
chloride (AsIII) in M9 buffer (NaCl 86 mM, Na
2
HPO
4
42 mM, KH
2
PO
4
22 mM and MgSO
4
1 mM), were then added to a nal concentration of
200 mM, 15.4 mM or 5 mM, respectively. Worms grown without oxidant
in medium with or without caramel served as additional controls. The
worms were scored every hour and they were considered as dead when
they did not move after repeated stimuli. Three independent experi-
ments were performed with more than 50 animals each.
2.2.2.3.3. Survival assays in moderate oxidative stress. Synchronized
young adults were incubated at 20 C in 96-well plates (approximately
10 worms /well) plus NGM containing none or 0.2% caramel solution
with FUdR (50
μ
M) and heat-killed bacteria. PQ was added to a nal
concentration of 4 mM. Control conditions contained none or 0.2%
caramel solution without PQ. The worms were scored every other day
and they were considered as dead when they did not move after repeated
stimuli. Animals were censored when they crawled off the well,
exploded or contained internally hatched larvae. Three independent
experiments were performed with more than 60 animals each.
2.2.2.3.4. Culture conditions and isolation of total RNA for gene
expression analysis. Day3 adult worms were grown 24 h at 20 C in 6-
well plates (approximately 1000 worms / well) containing NGM alone
or NGM plus 0,04 or 1% caramel solutions. All the media were
supplemented with FUdR (50
μ
M) and heat-killed bacteria. PQ, TBHP or
AsIII in M9 buffer were then added to a nal of 200 mM, 15.4 mM or
5 mM, respectively. Worms were incubated 2 h at 20 C in those oxida-
tive conditions before to be pelleted and frozen in liquid nitrogen. The
worms were then homogenized in Trizol (Ambion) with acid-washed
glass beads (425600 µm; B. Sigma-Aldrich) with Precellys®24 (Bertin
Technologies) at full speed, 4 times 10 s, 3 cycles. Total RNA was
extracted as previously described (Fradin et al., 2003).
2.2.2.3.5. Gene expression analysis. RNAs were treated with DNaseI
(Thermo Scientic) prior to be reverse transcribed to cDNA (High-ca-
pacity cDNA reverse transcription kits, Applied Biosystems). Quantita-
tive real-time PCR was performed using PowerUp
TM
SYBR® Green 2X
Mater Mix (Applied Biosystems). Gene expression levels were normal-
ized to pmp-3 and relative expression was calculated by the 2
-ΔΔCT
method. The relative expression of genes was analyzed by comparing
either the condition with oxidizing chemical to the same condition
without the chemical or the caramel conditions to the condition without
caramel. Primers are listed in Table S1.
2.3. Statistics
KRL test and TEAC assay: All experiments were performed in
duplicate (KRL) or triplicate (TEAC) and the results were expressed as
mean ±standard deviation (SD). Signicant differences (P <0.05) be-
tween the means were determined by the ANOVA test of software
Graphpad Prism.
Caenorhabditis elegans model: Kaplan-Meier survival curve and sta-
tistical analysis were generated by bioinformatics software GraphPad
Prism. Survival analysis and P values for gene expression are based on
log-rank and two-way ANOVA tests, respectively. P <0.05 was consid-
ered statistically signicant.
3. Results and discussion
For valid assessment, the antioxidant capacity of caramel samples
was determined using three complementary methods: TEAC/ABTS
assay, the KRLTest, and Caenorhabditis elegans model.
3.1. Results of the antioxidant capacity evaluated by in vitro assays
3.1.1. TEAC assay
3.1.1.1. Kinetic measurements. Among chemical assays, TEAC was rst
developed as a simple and convenient method for assessment of the total
antioxidant capacity. The rst step was to examine the kinetic behavior
of reactions between ABTS
.+
and caramels or common antioxidants.
Absorbance readings at 734 nm were taken during 20 min. Percentage of
inhibition versus time graphs are shown in Fig. 1.
Trolox and ascorbic acid reacted immediately with ABTS
.+
(Fig. 1A).
0
20
40
60
80
100
0 5 10 15 20
% inhibion
Reacng me (min)
18.8 μg trolox
15 μg Ascorbic acid
4 μg Gallic acid
A
0
20
40
60
80
100
0 5 10 15 20
% inhibion
Reacng me (min)
4 mg aromac caramel
0.4 mg Class I caramel colours
0.25 mg Class III caramel colours
0.5 mg Class IV caramel colours
B
Fig. 1. Rate of reaction of antioxidants with ABTS
.+
: A: common antioxidants - B: caramels.
C. Moretton et al.
Journal of Functional Foods 78 (2021) 104308
4
The inhibition percentage was constant over time (56.0 and 43.5% in-
hibition, respectively). Gallic acid had a fast initial rate but the end point
of inhibition was not reached after 20 min. Similar results of reaction
rates were obtained by Walker and Everette using Trolox, ascorbic acid
and plant polyphenols (Walker & Everette, 2009).
For aromatic caramel and caramel colours, the curves were compa-
rable to the gallic acid curve (Fig. 1B). In the same way, the end point
was not reached after 20 min. To compare different caramels, the
reacting time / color measurement was xed at 5 min as generally set in
previous publications (Alam et al., 2013; Amigoni et al., 2017; Seeram
et al., 2006).
3.1.1.2. TEAC of well-known antioxidants. As a quality control, the
TEAC value of two well-known antioxidants were determined. TEAC of
ascorbic acid was evaluated at 6.2+/-0.6 mmol Trolox equivalent/g, this
result was comparable to previous studies which reported mean values
between 4.6 and 6.0 mmol/g (Miller & Rice-Evans, 1997; Rice-Evans,
Miller, & Paganga, 1997; Walker & Everette, 2009).
TEAC of gallic acid was evaluated at 24.0+/-2mmol/g, this result
was also comparable to published studies indicating values between
15.3 and 34.4 mmol/g (Miller & Rice-Evans, 1997; Rice-Evans et al.,
1997; Walker & Everette, 2009).
3.1.1.3. TEAC of caramels. As shown in Fig. 2, all the caramels tested
exhibited antioxidant activity according to the TEAC test, between
20.5+/-1.7 for standard aromatic caramel to 401+/-30 µmol Trolox
equivalent /g for Class III caramel colour.
For caramels without ammonium or sulphite compounds (AC, BS,
CC-I, CC-II), the antioxidant capacity increases signicantly with EBC
colour. In the same way, inside Class III and IV caramel colours (CC-III,
CC-IV), the antioxidant capacity increases with colour intensity. Among
caramel colours, Class IV type showed lower antioxidant capacity and
Class III had the highest antioxidant activity. These results conrm those
obtained by Brenna et al. on commercial caramel colours using the FRAP
and DPPH* methods (Brenna et al., 2009).
To compare, chocolates had a TEAC value between 10 and 100 µmol
Trolox equivalent /g, dark chocolates showed a greater antioxidant ca-
pacity (Laliˇ
ci´
c-Petronijevi´
c, 2016; Todorovic et al., 2015). Honey had a
TEAC value between 0.3 and 8 µmol/g (Marylenlid et al., n.d.; Sancho
et al., 2016; Vit, 2009). Coffee was a well-known antioxidant. The TEAC
value of ground coffee had been reported between 100 and 300 µmol/g
(Brezov´
a, ˇ
Slebodov´
a, & Staˇ
sko, 2009; da Cruz et al., 2018).
3.1.2. Protection of red blood cells evaluated by the KRL test
TEAC test is a validated in vitro chemical method to evaluate the
overall antioxidant capacity of foods. But among limitations, the TEAC
assay has been challenged for its lack of biological relevance and lower
sensitivity compared to the ORAC test (Shahidi & Zhong, 2015). We
decided then to evaluate the antioxidant activity of caramels with a
biological test. For this method, whole blood is submitted to an oxidant
stress induced by free radical attack. Haemolysis is recorded by optical
density decay with the KRL microplate reader. The overall antioxidant
effectiveness of caramels in biological conditions was determined.
Main results of the KRLtest are presented in Fig. 3. We found that
all the caramel samples (aromatic, burnt sugar, class I, II, III, IV)
exhibited an antioxidant capacity from 308+/-1 to 5366+/-147 mg
GAEq/100 g.
As for the TEAC test, CC-III b had the highest antioxidant activity and
the antioxidant activity increases with the colour for each category of
caramels.
Other food matrices were previously analyzed with the same KRL
method. The results in wine were between 120 and 350 mg GAEq/100 g.
In coffee beverage and orange juice, the antioxidant activity has been
evaluated at 650700 and 7580 mg GAEq/100 g respectively (Unpub-
lished data from LARA-SPIRAL and Prost et al., 2017).
3.1.3. Comparison of results between methods and with other foods
To check the reliability of our data, the results obtained with the 8
marketed caramel samples using TEAC and KRL-test were compared.
Fig. 4 shows a strong linear correlation between the two sets of results
(r =0.966). Both methods, one rather chemical and one rather biolog-
ical, conrm the antioxidant potential of caramels.
The antioxidant effects of caramels are equivalent or superior to
those obtained with the same measurement techniques in other food
products recognized as antioxidant. In Europe, the combined mean ex-
posures to the four caramel colours (E 150a-150d) was evaluated from
15 to 57 mg/kg bw/day for adults, which could be equivalent to 14 g
for a weight of 70 kg (EFSA, 2012). Based on our results, the antioxidant
capacity from the daily ingestion of caramel colours is almost equivalent
to a glass of orange juice (140 vs. 160 mg gallic acid equivalent for fruit
juice) (Unpublished data from LARA-SPIRAL, Debabbi et al., 2017). The
exposure to caramel colours would reach 41165 mg/kg bw/day among
the highest consumers (95th percentile), the equivalent of a 311 g daily
intake. This consumption could be compared to a cup of coffee in term of
antioxidant activity. The consumption of aromatic caramels is more
difcult to assess but they also contribute to the exposure. Even when
related to its level of consumption, caramels could consequently be a
signicant contributor to the daily intake of antioxidant compounds. As
an interesting point about the risk/benet balance, it should be noted
that the level of consumption recorded in Europe remains below the
group acceptable daily intake applicable to combined four colours
(300 mg/kg bw/day (EFSA, 2012)).
a
20.5
±1.7
b
95
±6
c
262
±24
c
280
±17
c
290
±13
d
401
±30
b
76
±6
e
161
±12
0
100
200
300
400
500
AC
EBC
570
BS
EBC
11 000
CC-I
EBC
17 000
CC-II
EBC
19 000
CC-III a
EBC
22 000
CC-III b
EBC
35 000
CC-IV a
EBC
21 000
CC-IV b
EBC
46 000
Fig. 2. Antioxidant activity of caramel samples by TEAC test data are expressed
in Trolox equivalent (µmol/g of sample) and presented as the means ±standard
deviation. Bars with no letters in common are signicantly different (p <0.05,
ANOVA test).
a
308
±1
b
1,865
±33
c
3,667
±17
d
4,489
±20
e
4,079
±19
f
5,366
±147
b
2,067
±21
g
3,266
±87
0
1000
2000
3000
4000
5000
6000
AC
EBC
570
BS
EBC
11 000
CC-I
EBC
17 000
CC-II
EBC
19 000
CC-III a
EBC
22 000
CC-III b
EBC
35 000
CC-IV a
EBC
21 000
CC-IV b
EBC
46 000
Fig. 3. Antioxidant activity of caramel samples by KRL test data are
expressed in gallic acid equivalent (mg/100 g caramel) and presented as the
means ±standard deviation (SD). Bars with no letters in common are signi-
cantly different (p <0.05, ANOVA test).
C. Moretton et al.
Journal of Functional Foods 78 (2021) 104308
5
3.2. Effect of caramels on survival and gene expression in C. Elegans
model
Four samples of the eight tested for KRL and TEAC studies were
selected for our investigation on C. elegans. Thus, we performed analyses
with aromatic caramel, burnt sugar and caramels colours class III and
class IV, the latters showing the highest colour intensity (EBC 35 000
and 46 000, respectively).
3.2.1. Caramel solutions increase survival of C. Elegans in response to
severe oxidative conditions
In order to highlight any direct or indirect antioxidant property of
caramels, we tested the resistance of worms to different oxidative
stresses in the presence of caramel solutions. We scored the worms
survival during treatment with different chemicals at a concentration
that generate severe oxidative stress.
Under physiological condition, the different concentrations of cara-
mels didnt decrease the wormssurvival, suggesting that no caramel
has a toxic effect on C. elegans (Fig. 5). Consistent with this nding, non-
toxicity of class IV caramels had been highlighted using in vitro assays
0
1000
2000
3000
4000
5000
6000
0 50 100 150 200 250 300 350 400 450
KRL (GAEq mg/100g)
TEAC (μmol Trol equivalent /g)
r = 0.966
Fig. 4. Comparison of results of the antioxidant activity obtained with the TEAC method (µmol Trolox equivalent/g of sample) and the KRL method (mg GAEq/100 g
of sample).
Fig. 5. Survival of worms to severe oxidative stresses in presence of caramels. Worms were pretreated with none (0%), 0.04%, 0.2% or 1% of caramel AC, caramel
BS, caramel CC-IIIb or caramel CC-IVb before addition of oxidizing chemicals: paraquat/PQ (A), tert-Butyl hydroperoxide/TBHP (B) or trivalent Arsenic/AsIII (C).
Worms incubated without oxidant in medium with or without caramel served as control (Ctrls): both survival curves are superimposed at 100% over the experi-
mentation time. **** p <0.0001, *** p <0.001, **p <0.01 and *p <0.05 compared to control survival.
C. Moretton et al.
Journal of Functional Foods 78 (2021) 104308
6
and Drosophila melanogaster model (Mateo-Fern´
andez et al., 2019). On
the contrary, the different concentrations of caramels, whatever their
nature, increased worm survival under the 3 oxidizing environments
(PQ, TBHP and AsIII), the level of protection depends on the type of
oxidative stress induced (Fig. 5).
PQ generates superoxide radicals after reduction in the mitochon-
dria. No worm survived a 12-hour exposure to PQ without caramel.
Caramels AC and CC-IVb showed higher resistance to PQ than caramels
BS and CC-IIIb with more than 40% live worms after 18 h of treatment
(Fig. 5A). The 0.2% concentration of caramel appeared to be the optimal
concentration for PQ resistance.
TBHP is a strong oxidant, often more effective than hydrogen
peroxide. All worms were killed after 10-hours treatment with TBHP
without caramel (Fig. 5B). Although survival curves are signicantly
increased with the different caramels, the median survival of worms to
TBHP is not changed with any of the different caramel solutions. By
contrast, the maximum survival of worms at treatment is increased in
the presence of the caramels. Whatever their concentrations, caramels
BS, CC-IIIb and CC-IVb had the most positive effect.
Results similar to TBHP were obtained after treatment of worms with
AsIII (Fig. 5C).
The antioxidant effects of each caramel in vivo depend on the
oxidizing conditions tested, preventing the classication of the different
caramels according to an antioxidant activity. It is therefore difcult to
compare these results with those obtained with in vitro tests. In addition,
the possible metabolism of caramels by the worm can certainly modify
their antioxidant properties. The results show that caramels of class III
and IV have an antioxidant effect in vivo.
3.2.2. Caramels induce resistance to moderate paraquat exposure
Knowing the positive effect of caramels on severe PQ exposure, we
investigated the resistance of worms to moderate PQ exposure (4 mM) in
presence of the caramel solutions. The caramels were used at the optimal
concentration of survival in severe oxidative conditions: 0.2%. Under
physiological condition, caramels did not decrease the wormslifespan,
suggesting that no caramel has a toxic effect on C. elegans (Ctrl vs AC/
BS/CC on Fig. 6). On the contrary, caramel AC signicantly increased
the longevity of the worms.
In moderate oxidative conditions and without caramels, the
longevity of worms logically decreased (Ctrl vs Ctrl +PQ, Fig. 6). With
caramels, the longevity of worms has not changed for burnt sugar,
whereas the 3 other caramels signicantly increased the lifespan of
worms under this oxidizing condition (Ctrl +PQ vs AC/BS/CC +PQ,
Fig. 6).
These results conrm the benecial effect of those caramels to pre-
vent damage caused by oxidants. The result with aromatic caramel
suggests that this caramel has a positive effect on the longevity of worms
which is independent of oxidative stress and/or which is partly depen-
dent on physiological oxidative stress. Worms incubated with caramels
CC-IIIb and CC-IVb have the same longevity whether they are incubated
with or without PQ, showing that these caramels protect the worms from
induced moderate oxidative stress.
Although surprising, the results obtained with burnt sugar show that
the antioxidant properties of any molecule is conditioned by various
factors including oxidative stress itself. Caramel BS either has oxidative
properties which deteriorate over time or is metabolized by the worm,
inducing an antioxidant effect in the short term (against severe stress)
and not in the long term (against moderate stress). Whatever the cause,
this result reinforces the antioxidant effect of the 3 other caramels tested
against moderate oxidative stress which is induced by PQ.
3.2.3. Expression of genes coding response to severe oxidative stress is
partially modulated by caramels
We further explored the antioxidant effects of the caramel solutions.
Fig. 6. Effects of caramels on lifespan of C. elegans in moderate oxidative stress. Young adults of N2 strain were grown on NGM plates containing none or 4 mM
paraquat/PQ with no caramel (Ctrl) or 0.2% caramel AC (A), caramel BS (B), caramel CC-IIIb (C) or caramel CC-IVb (D) solutions. *** p <0.001: Ctrl vs Ctrl +PQ.
∫ ∫ ∫ ∫ p <0.0001 and p <0.05: caramel vs caramel +PQ. ▴▴▴▴ p <0.0001 and p <0.05: caramel vs Ctrl. #### p <0.0001 and ### p <0.001: caramel +PQ
vs Ctrl +PQ.
C. Moretton et al.
Journal of Functional Foods 78 (2021) 104308
7
We veried if caramels could directly or indirectly modulate gene
expression of enzymes involved in antioxidant response of C. elegans.
Severe oxidative stress was then administered to the worms with the 3
oxidizing substances for 2 h with or without pretreatment with the
caramel solutions.
Targeted antioxidant response was analyzed with the measurement
of the relative expression of genes encoding some worms key antioxi-
dant enzymes: superoxide dismutase SOD-3 and glutathione-S-
transferases GST-4 and GST-14 (40). GSTs have an important role in
antioxidant recycling but also deactivate electrophilic xenobiotics or
metabolites. Expression of the gene encoding glutamate-cysteine ligase
GCS-1, an important enzyme for glutathione synthesis, was also
measured (Ferguson & Bridge, 2019).
3.2.3.1. Modulation of oxidative stress response by severe oxidative stress
or caramels. In the absence of caramel, exposure of worms with each
oxidizing chemical induced the expression of genes coding for the
antioxidant enzymes apart from sod-3 whose expression is not enhanced
by TBHP and AsIII (Control bars on Figs. 7 and S1), which is in line with
the oxidizing species generated and their subcellular localization.
In a non-oxidative condition, the caramels did not inuence the
expression of the genes encoding the antioxidant enzymes (Fig. S2).
These results suggest that caramels do not directly regulate the expres-
sion of these genes. Although non-signicant, the highest concentration
of caramel CC-IVb down-regulated expression of the genes sod-3, gst-4
and gst-14, implying a possible effect of this caramel on the level of
physiological stress of the worms.
Fig. 7. Relative expression of antioxidant genes induced by severe oxidative stress in presence of caramels. Worms were pretreated with none (Ctrl) or 0.04% (A) or
1% (B) of caramel AC, caramel BS, caramel CC-IIIb or caramel CC-IVb before addition of 200 mM paraquat/PQ. After 2 h of PQ exposure, relative expression of genes
was analyzed by comparing the condition with caramel to the Ctrl condition. The pmp-3 gene was used to normalize levels of gene expression. *p <0.05 (caramel
vs Ctrl).
C. Moretton et al.
Journal of Functional Foods 78 (2021) 104308
8
3.2.3.2. Modulation of oxidative stress response by caramels in severe
oxidative stress. The caramels studied had very little effect on the tran-
scriptional response induced after TBHP and AsIII treatments (Figs. S3
and S4) while they modulated the antioxidant response induced by PQ
exposure (Fig. 7). PQ-induced sod-3 expression was inhibited by at least
one concentration of aromatic caramel, burnt sugar and class III and
class IV caramels colours, suggesting an inhibition or a blocking of su-
peroxide anion by the caramels. On the other hand, the genes coding for
GST-4 and GST-14 were more expressed in the presence of the 4 cara-
mels used at the lowest concentration (0.04%, Fig. 7A) while the highest
concentration of caramels (1%, Fig. 7B) had no effect or decreased non-
signicantly the expression of these genes (caramel AC and CC-IIIb for
the expression of gst-14). These results strongly suggest a direct effect of
caramels on superoxide anions but also on the production of antioxidant
recycling dependent on GST activities.
Induction of the gst genes shows that 0.04% caramels have a partial
antioxidant effect. It is known that the expression of GSTs can also be
regulated independently of oxidative stress (e.g. hormones, xenobi-
otics). We cannot exclude such a regulation, but it would be partly
dependent on oxidative stress since caramels do not induce the expres-
sion of these genes under physiological condition (Fig. S2). Caramels
seem to have no effect on the upstream processes of antioxidant re-
actions because they had no signicant effect on the expression of gcs-1
whatever the induced oxidative stress (Figs. 7, S3 and S4).
Different studies have described the benecial effects of different
food compounds on different biological functions of C. elegans including
the maintenance of protein homeostasis, mobility and longevity. These
compounds such as coffee and orange extracts and thioallyls enhance
the oxidative stress resistance of the worms (Amigoni et al., 2017;
Dostal, Roberts, & Link, 2010; Ogawa, Kodera, Hirata, Blackwell, &
Mizunuma, 2016; Wang et al., 2020). In the present study, we show that,
caramels are also able to improve the resistance of worms against
oxidative stress, certainly thanks to the antioxidant properties demon-
strated by in vitro tests. A future study will demonstrate whether these
additives have other properties on the biological functions of C. elegans.
3.3. Conclusion
Three different methods used to evaluate the antioxidant activity of
marketed aromatic caramels and caramel colours showed promising
results that strongly support the hypothesis that the process of sugars
heating leads to compounds with benecial antioxidant properties.
In vitro TEAC and KRL assays have demonstrated that the antioxidant
activity depends on the colour of the product and the caramelization
promoters used. As caramels are metabolized in vivo, it therefore was
essential to validate that this antioxidant effect is preserved in vivo.
C. elegans is considered as a relevant animal model to evaluate the in vivo
antioxidant effects of bioactive compounds (Ayuda-Dur´
an, Gonz´
alez-
Manzano, Gonz´
alez-Param´
as, & Santos-Buelga, 2020). Results in this
model conrmed that caramels are able to prevent damage caused by
oxidative stress and this activity strongly depends on the dose and type
of caramel tested.
The structure of most molecules in caramels remains actually un-
known but the characterization of antioxidant molecules present in
products obtained by the heat treatment of sugars would probably be a
next topic of interest (Rodríguez et al., 2019). They probably differ
depending on the reactants used and level of heating process. A better
knowledge of their content and chemical composition in caramels would
partly explain the different antioxidant properties observed in our study.
Our results open up a promising eld of research on the antioxidant
potential of heated-formed compounds present in caramels and bring
new elements that highlight the potential benecial health effects of
caramels. From a more prospective point of view and with the support of
additional studies, caramels could represent a signicative source of
antioxidants that may prevent the effects of free radical-induced phys-
iological stages or diseases. Future applications would particularly be of
great interest in aging prevention, clinical or sports nutrition.
Ethics statement
We have read and adhered to the Publishing Ethics regarding animal
experiments.
CRediT authorship contribution statement
C´
edric Moretton: Investigation, Visualization, Writing - original
draft. C´
ecile Gouttefangeas: Investigation, Supervision. Constance
Dubois: Investigation. Fr´
ed´
eric Jacques Tessier: Investigation,
Writing - review & editing. Chantal Fradin: Investigation, Writing -
review & editing. Emmanuelle Prost-Camus: Investigation, Writing -
review & editing. Michel Prost: Investigation, Writing - review &
editing. Marc Haumont: Investigation, Writing - review & editing.
Henri Nigay: Supervision, Resources.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgements
We thank the Caenorhabditis Genetics Center for providing the
C. elegans N2 wild-type and bacterial OP50 strain and Nutrizz company
for their editorial assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jff.2020.104308.
References
Alam, Md. N., Bristi, N. J., & Raquzzaman, Md. (2013). Review on in vivo and in vitro
methods evaluation of antioxidant activity. Saudi Pharmaceutical Journal, 21(2),
143152. https://doi.org/10.1016/j.jsps.2012.05.002.
Amigoni, L., Stuknyt˙
e, M., Ciaramelli, C., Magoni, C., Bruni, I., De Noni, I., et al. (2017).
Green coffee extract enhances oxidative stress resistance and delays aging in
Caenorhabditis elegans. Journal of Functional Foods, 33, 297306. https://doi.org/
10.1016/j.jff.2017.03.056.
Arribas, B., Su´
arez-Pereira, E., Ortiz Mellet, C., García Fern´
andez, J. M., Buttersack, C.,
Rodríguez-Cabezas, M. E., et al. (2010). Di-D-fructose dianhydride-enriched
caramels: Effect on colon microbiota, inammation, and tissue damage in
trinitrobenzenesulfonic acid-induced colitic rats. Journal of Agricultural Food
Chemistry, 58(10), 64766484. https://doi.org/10.1021/jf100513j.
Ayuda-Dur´
an, B., Gonz´
alez-Manzano, S., Gonz´
alez-Param´
as, A. M., & Santos-Buelga, C.
(2020). Caenorhabditis elegans as a Model Organism to Evaluate the Antioxidant
Effects of Phytochemicals. Molecules, 25(14). https://doi.org/10.3390/
molecules25143194.
Brahmi, F., Nury, T., Debbabi, M., Hadj-Ahmed, S., Zarrouk, A., Prost, M., et al. (2018).
Evaluation of Antioxidant, Anti-Inammatory and Cytoprotective Properties of
Ethanolic Mint Extracts from Algeria on 7-Ketocholesterol-Treated Murine RAW
264.7 Macrophages. Antioxidants, 7(12), 184. https://doi.org/10.3390/
antiox7120184.
Brenna, O. V., Ceppi, E. L. M., & Giovanelli, G. (2009). Antioxidant capacity of some
caramel-containing soft drinks. Food Chemistry, 115(1), 119123. https://doi.org/
10.1016/j.foodchem.2008.11.059.
Brezov´
a, V., ˇ
Slebodov´
a, A., & Staˇ
sko, A. (2009). Coffee as a source of antioxidants: An
EPR study. Food Chemistry, 114(3), 859868. https://doi.org/10.1016/j.
foodchem.2008.10.025.
Cases, J., Romain, C., Marín-Pag´
an, C., Chung, L. H., Rubio-P´
erez, J. M., Laurent, C.,
et al. (2017). Supplementation with a Polyphenol-Rich Extract, PerfLoad®, Improves
Physical Performance during High-Intensity Exercise: A Randomized, Double Blind,
Crossover Trial. Nutrients, 9(4). https://doi.org/10.3390/nu9040421.
Cruz, R. G. da, Vieira, T. M. F. de S., Lira, S. P. de, Cruz, R. G. da, Vieira, T. M. F. de S., &
Lira, S. P. de. (2018). Potential antioxidant of brazilian coffee from the region of
Cerrado. Food Science and Technology, 38(3), 447453. 10.1590/1678-457x.08017.
Debabbi et al. (2017). Etude comparative des prols anti-oxydants et de la composition
en polyph´
enols de fruits et de l´
egumes associ´
es au r´
egime m´
editerran´
een. In Norbert
Latruffe, Vin, nutrition m´
editerran´
eenne et sant´
e, une association vertueuse. https
C. Moretton et al.
Journal of Functional Foods 78 (2021) 104308
9
://eud.u-bourgogne.fr/sciences/521-vin-nutrition-mediterraneenne-et-sante-9782
364411999.html.
Dostal, V., Roberts, C. M., & Link, C. D. (2010). Genetic mechanisms of coffee extract
protection in a Caenorhabditis elegans model of β-amyloid peptide toxicity. Genetics,
186(3), 857866. https://doi.org/10.1534/genetics.110.120436.
EFSA. (2012). Rened exposure assessment for caramel colours (E 150a, c, d). EFSA
Journal, 10(12), 3030. https://doi.org/10.2903/j.efsa.2012.3030.
Ferguson, G. D., & Bridge, W. J. (2019). The glutathione system and the related thiol
network in Caenorhabditis elegans. Redox Biology, 24, Article 101171. https://doi.
org/10.1016/j.redox.2019.101171.
Finley, J. W., Kong, A.-N., Hintze, K. J., & Je, E. H. (2011). Antioxidants in Foods: State of
the Science Important to the Food Industry. Journal of Agricultural and Food
Chemistry, 10.
Fradin, C., Kretschmar, M., Nichterlein, T., Gaillardin, C., DEnfert, C., & Hube, B.
(2003). Stage-specic gene expression of Candida albicans in human blood.
Molecular Microbiology, 47(6), 15231543. https://doi.org/10.1046/j.1365-
2958.2003.03396.x.
Golon, A., & Kuhnert, N. (2012). Unraveling the chemical composition of caramel.
Journal of Agricultural Food Chemistry, 60(12), 32663274. https://doi.org/10.1021/
jf204807z.
Laliˇ
ci´
c-Petronijevi´
c, J. (2016). Effect of Chocolate Storage on Total Phenolics, Flavan-3-
Ols, Proanthocyanidins, Oxidative Stability and Antioxidant Capacity. Food
Technology and Biotechnology, 54(1). 10.17113/ftb.54.01.16.4014.
Licht, B. H., Shaw, K., Smith, C., Mendoza, M., Orr, J., & Myers, D. V. (1992).
Characterization of Caramel Colour IV. Food and Chemical Toxicology, 30(5),
365373. https://doi.org/10.1016/0278-6915(92)90062-p.
Marylenlid, I., Atilio, C., Lorena, D., Mariana, E., & Patricia, V. (n.d.). 19. Cosmetic
properties of honey. 1. Antioxidant activity. 8.
Mateo-Fern´
andez, M., Alves-Martínez, P., Del Río-Celestino, M., Font, R., Merinas-
Amo, T., & Alonso-Moraga, ´
A. (2019). Food Safety and Nutraceutical Potential of
Caramel Colour Class IV Using In Vivo and In Vitro Assays. Foods (Basel,
Switzerland), 8(9). https://doi.org/10.3390/foods8090392.
Miller, N. J., & Rice-Evans, C. A. (1997). The relative contributions of ascorbic acid and
phenolic antioxidants to the total antioxidant activity of orange and apple fruit juices
and blackcurrant drink. Food Chemistry, 60(3), 331337. https://doi.org/10.1016/
S0308-8146(96)00339-1.
Moretton, C. (2009). Analyse des caramels liquides: D´
eveloppement et validation de
nouvelles m´
ethodes bas´
ees sur la chromatographie en phase liquide
bidimensionnelle (LC-LC) [PhD Thesis]. http://www.theses.fr/2009LYO10279
/document.
Ogawa, T., Kodera, Y., Hirata, D., Blackwell, T. K., & Mizunuma, M. (2016). Natural
thioallyl compounds increase oxidative stress resistance and lifespan in
Caenorhabditis elegans by modulating SKN-1/Nrf. Scientic Reports, 6(1), 21611.
https://doi.org/10.1038/srep21611.
Orban, J. I., Patterson, J. A., Sutton, A. L., & Richards, G. N. (1997). Effect of sucrose
thermal oligosaccharide caramel, dietary vitamin-mineral level, and brooding
temperature on growth and intestinal bacterial populations of broiler chickens.
Poultry Science, 76(3), 482490. https://doi.org/10.1093/ps/76.3.482.
Peinado, M. J., Ech´
avarri, A., Ruiz, R., Su´
arez-Pereira, E., Ortiz Mellet, C., García
Fern´
andez, J. M., et al. (2013). Effects of inulin and di-D-fructose dianhydride-
enriched caramels on intestinal microbiota composition and performance of broiler
chickens. Animal, 7(11), 17791788. https://doi.org/10.1017/
S1751731113001572.
Phan-Thi, H., Durand, P., Prost, M., Prost, E., & Wach´
e, Y. (2016). Effect of heat-
processing on the antioxidant and prooxidant activities of β-carotene from natural
and synthetic origins on red blood cells. Food Chemistry, 190, 11371144. https://
doi.org/10.1016/j.foodchem.2015.06.088.
Pisoschi, A. M., & Negulescu, G. P. (2012). Methods for Total Antioxidant Activity
Determination: A Review. Biochemistry & Analytical Biochemistry, 01(01). https://
doi.org/10.4172/2161-1009.1000106.
Prost et al. (2017). Colloque sous les auspices de la Chaire UNESCO, culture et tradition
du vin. In Norbert Latruffe, Vin, nutrition m´
editerran´
eenne et sant´
e, une association
vertueuse. https://eud.u-bourgogne.fr/sciences/521-vin-nutrition-mediterraneenne
-et-sante-9782364411999.html.
Prost, M. (1992). Process for the determination by means of free radicals of the
antioxidant properties of a living organism or a potentially aggressive age (United
States Patent No. US5135850A). https://patents.google.com/patent/US5135850A/
en.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Free Radical Biology & Medicine, 26(910), 12311237. https://doi.org/10.1016/
s0891-5849(98)00315-3.
Rice-Evans, C., Miller, N., & Paganga, G. (1997). Antioxidant properties of phenolic
compounds. Trends in Plant Science, 2(4), 152159. https://doi.org/10.1016/S1360-
1385(97)01018-2.
Rodríguez, A., Lema, P., Bessio, M. I., Moyna, G., Panizzolo, L. A., & Ferreira, F. (2019).
Isolation and Characterization of Melanoidins from Dulce de Leche. A Confectionary
Dairy Product. Molecules, 24(22), 4163. https://doi.org/10.3390/
molecules24224163.
Rubio, L. A., Peinado, M. J., Ech´
avarri, A., Ruiz, R., Su´
arez-Pereira, E., Mellet, C. O., et al.
(2014). A Di-D-Fructose Dianhydride-Enriched Caramel Modulates Pig Fecal
Microbiota Composition. AiM, 04(5), 242251. https://doi.org/10.4236/
aim.2014.45031.
Saito, K., & Tomita, F. (2000). Difructose anhydrides: Their mass-production and
physiological functions. Bioscience, Biotechnology, and Biochemistry, 64(7),
13211327. https://doi.org/10.1271/bbb.64.1321.
Sancho, M. T., Pascual-Mat´
e, A., Rodríguez-Morales, E. G., Os´
es, S. M., Escriche, I.,
Periche, ´
A., et al. (2016). Critical assessment of antioxidant-related parameters of
honey. International Journal of Food Science & Technology, 51(1), 3036. https://doi.
org/10.1111/ijfs.12988.
Seeram, N. P., Henning, S. M., Niu, Y., Lee, R., Scheuller, H. S., & Heber, D. (2006).
Catechin and caffeine content of green tea dietary supplements and correlation with
antioxidant capacity. Journal of Agricultural and Food Chemistry, 54(5), 15991603.
https://doi.org/10.1021/jf052857r.
Sengar, G., & Sharma, H. K. (2014). Food caramels: A review. Journal of Food Science and
Technology, 51(9), 16861696. https://doi.org/10.1007/s13197-012-0633-z.
Shahidi, F., & Zhong, Y. (2015). Measurement of antioxidant activity. Journal of
Functional Foods, 18, 757781. https://doi.org/10.1016/j.jff.2015.01.047.
Todorovic, V., Redovnikovic, I. R., Todorovic, Z., Jankovic, G., Dodevska, M., &
Sobajic, S. (2015). Polyphenols, methylxanthines, and antioxidant capacity of
chocolates produced in Serbia. Journal of Food Composition and Analysis, 41,
137143. https://doi.org/10.1016/j.jfca.2015.01.018.
Tsai, P.-J., Yu, T.-Y., Chen, S.-H., Liu, C.-C., & Sun, Y.-F. (2009). Interactive role of color
and antioxidant capacity in caramels. Food Research International. http://agris.fao.or
g/agris-search/search.do?recordID=US201301623845.
Vit, P. (2009). Expanded parameters to assess the quality of honey from Venezuelan bees
(Apis melllifera). Journal of ApiProduct and ApiMedical Science, 1(3), 7281. https://
doi.org/10.3896/IBRA.4.01.3.03.
Walker, R. B., & Everette, J. D. (2009). Comparative reaction rates of various
antioxidants with ABTS radical cation. Journal of Agricultural and Food Chemistry, 57
(4), 11561161. https://doi.org/10.1021/jf8026765.
Wang, J., Deng, N., Wang, H., Li, T., Chen, L., Zheng, B., et al. (2020). Effects of Orange
Extracts on Longevity, Healthspan, and Stress Resistance in Caenorhabditis elegans.
Molecules, 25(2), 351. https://doi.org/10.3390/molecules25020351.
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