Article
pubs.acs.org/JAFC
Influence of Steviol Glycosides on the Stability of Vitamin C and
Anthocyanins
Łukasz Woźniak,* Krystian Marszałek, and Sylwia Skąpska
Department of Fruit and Vegetable Product Technology, Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology,
36 Rakowiecka Street, 02-532 Warsaw, Poland
S Supporting Information
*
ABSTRACT: A high level of sweetness and health-promoting properties make steviol glycosides an interesting alternative to
sugars or artificial sweeteners. The radical oxygen species scavenging activity of these compounds may influence the stability of
labile particles present in food. Model buffer solutions containing steviol glycosides, a selected food antioxidant (vitamin C or
anthocyanins), and preservative were analyzed during storage. The addition of steviol glycosides at concentrations of 50, 125, and
200 mg/L increased the stability of both ascorbic and dehydroascorbic acid (degradation rates decreased up to 3.4- and 4.5-fold,
respectively); the effect was intensified by higher sweetener concentrations and higher acidity of the solutions. Glycosides used
alone did not affect the stability of anthocyanins; however, they enhanced the protective effect of sugars; half-life times increased
by ca. 33% in the presence of sucrose (100 g/L) and by ca. 52% when both sucrose (100 g/L) and glycosides (total 200 mg/L)
were used. Steviol glycosides concentrations remained stable during experiments.
KEYWORDS: anthocyanins, Stevia rebaudiana, steviol glycosides, vitamin C
■
INTRODUCTION
Stevia (Stevia rebaudiana Bertoni) is a perennial shrub native to
Paraguay and Brazil. One of the major constituents of its leaves
is a group of ent-kaurene diterpenoid glycosides commonly
referred to as steviol glycosides. Their content varies depending
on cultivation conditions, usually accounting for 4−20% of the
leaf dry weight.1 Stevioside and rebaudioside A constitute about
90% of the total glycosides mass; apart from them, over 30 of
these compounds were identified in S. rebaudiana leaves.2
From a practical point of view the most important property
of steviol glycosides is their highly sweet taste (approximately
200−300 times sweeter than sucrose). The leaves of S.
rebaudiana have traditionally been used for hundreds of years
by Guarani Indians, but their application in Western diets is
relatively new.3 Crude stevia extracts were first commercialized
in Japan in the early 1970s, and since then they have gained
popularity in East Asian and Latin American countries.4 The
United States, Australia, New Zealand, and members of the
European Union only very recently joined the list of countries
authorizing the use of steviol glycosides; however, many
consumers in those areas have already found stevia to be an
interesting natural sweetener.
Gastric juices and digestive enzymes from humans and
animals are unable to degrade steviol glycosides. In addition,
their high molecular masses along with hydrophilic character
make their absorption in the intestines very unlikely; however,
intestinal microbiota are able to convert glycosides into
saccharides and an aglycone (steviol), which can penetrate
into the bloodstream. Absorbed steviol is converted into steviol
glucuronide in the liver and then excreted via the urinary and
biliary tract.5
Besides their sweetening properties, steviol glycosides and
related compounds offer therapeutic benefits including antiinflammatory, antiviral, antitumor, antihyperglycemic, antihy© 2014 American Chemical Society
pertensive, antidiarrheal, diuretic, and immunomodulatory
effects.5,6 Some of the above-mentioned activities arise from
the antioxidant properties of steviol glycosides and their
metabolites. Studies conducted by Stoyanova et al.7 and
Geuns et al.8 revealed that sweeteners from S. rebaudiana are
very efficient reactive oxygen species scavengers: half-inhibitory
concentrations on hydroxyl (HO•) and superoxide (O2•−)
radicals were approximately 0.2 and 0.3 mM, respectively. The
antioxidant properties of steviol glycosides suggest that these
compounds might influence the quality of foods by delaying
degradation of their labile ingredients such as ascorbic acid or
phenolic compounds.
Studies on the interactions between glycosides from S.
rebaudiana and other food constituents are very scarce. Kroyer
investigated the interactions in binary model solutions
containing stevioside and water-soluble vitamins (ascorbic
acid, thiamin, riboflavin, pyridoxine, and nicotinic acid) or
low-calorie sweeteners (saccharin, cyclamate, aspartame,
acesulfame, and neohesperidin dihydrochalcone).9,10 All
samples were incubated in the dark, at a temperature of 80
°C for up to 4 h. The majority of the trials showed no
significant change in the concentrations of the examined
compounds; however, the protective effect of stevioside was
observed in the case of the degradation of ascorbic acid. Its
stability in the presence of glycoside (27% of ascorbic acid
remained after 4 h) was significantly higher compared to that of
the control sample (13% remained after 4 h).
The stability of steviol glycosides in various foodstuffs was
investigated by several research teams. Selected matrices were
Received:
Revised:
Accepted:
Published:
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August 26, 2014
October 28, 2014
October 29, 2014
October 29, 2014
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Journal of Agricultural and Food Chemistry
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Table 1. Composition of Model Solutions To Study the Interactions between Steviol Glycosides and Anthocyaninsa
symbol
C3G (mg L−1)
C3G/C
C3G/Ste200
C3G/Ste125
C3G/Ste50
C3G/RebA200
C3G/RebA125
C3G/RebA50
C3G/Suc
C3G/GF
C3G/Mix
P3G/C
P3G/Ste200
P3G/Ste125
P3G/Ste50
P3G/RebA200
P3G/RebA125
P3G/RebA50
P3G/Suc
P3G/GF
P3G/Mix
100
100
100
100
100
100
100
100
100
100
P3G (mg L−1)
Ste (mg L−1)
RebA (mg L−1)
Suc (g L−1)
Glu (g L−1)
Fru (g L−1)
200
125
50
200
125
50
100
100
100
100
100
100
100
100
100
100
100
100
100
50
50
50
50
100
200
125
50
200
125
50
100
100
100
100
BA (mg L−1)
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
a
The acidity of all solutions was pH 3.6 and was set using citric acid−sodium phosphate buffer prepared according to the method of Ruzin.20
Abbreviations: BA, benzoic acid; C3G, cyanidin-3-glucoside; Fru, fructose; Glu, glucose; P3G, pelargonidin-3-glucoside; RebA, rebaudioside A; Ste,
stevioside; Suc, sucrose.
Waters) was used. Separation of the 10 μL samples was performed
within 12 min at a flow rate of 1 mL min−1 and a column temperature
of 40 °C. Samples were eluted using a gradient of 1.8 mM
hydrochloric acid (solvent A) and acetonitrile (solvent B), as follows
(time, corresponding composition): 0 min, 65% A; 3 min, 65% A; 5
min, 60% A; 7 min, 60% A; 9 min, 65% A; 12 min, 65% A.
Compounds were quantified using UV absorption at 210 nm.
The L-ascorbic (AA) and L-dehydroascorbic acid (DHAA) contents
were measured according to the a method proposed by OdriozolaSerrano et al.18 Before analysis, 1 mL of the sample was diluted with 1
mL of 0.01% phosphoric acid (determination of AA) or 1 mL of 1 g
L−1 solution of dithiothreitol (DTT) in 0.01% phosphoric acid
(determination of AA and DHAA sum). The samples with DTT were
kept in the cold (4 °C) for at least 1 h before injection. Separation was
done on a Sunfire C18, 5 μm, 4.6 mm × 250 mm analytical column
with a Sunfire C18 Sentry guard cartridge, 5 μm, 4.6 mm × 20 mm
(both Waters). Analysis of the 10 μL samples was performed within 10
min at a flow rate 1 mL min−1 and a column temperature of 25 °C.
The samples were eluted isocratically using 0.01% phosphoric acid.
The compounds were quantified using UV absorption at 245 nm.
Anthocyanin concentrations were determined using a method
proposed by Oszmiański19 using the same analytical column and guard
column as described above. Separation of the 10 μL samples was
performed within 26 min at a flow rate of 1 mL min−1 and a column
temperature of 25 °C. The samples were eluted using a gradient of
80% solution of acetonitrile in 4.5% formic acid (solvent A) and 4.5%
formic acid (solvent B), as follows (time, corresponding composition):
0 min, 0% A; 7 min, 15% A; 15 min, 15% A; 21 min, 100% A; 26 min,
0% A. Compounds were quantified using vis absorption at 520 nm.
Glucose, fructose, and sucrose were determined according to EN
12630:1999 standard. For the analysis a Sugar-Pak I, 10 μm, 6.5 mm ×
300 mm analytical column with a Sugar-Pak Guard-Pak insert, 10 μm
(both Waters), was used. Separation of the 10 μL samples was
performed within 18 min at a flow rate 0.5 mL min−1 and a column
temperature of 90 °C. Samples were eluted isocratically using 0.1 mM
calcium disodium EDTA. The compounds were quantified using a
refractive index detector.
Model Solutions. During research work, two series of experiments
were conducted. The first investigated the interactions between steviol
glycosides and two anthocyanins, cyanidin-3-glucoside and pelargoni-
carbonated drinks,11−14 dairy products and pastries,15,16 and
hot beverages, model solutions, and solid sweetener.9,10 All of
the cited publications show that the stability of glycosides
decreases with increasing acidity (significant decomposition at
room temperature occurred only in solutions with a pH <2).
The thermal degradation of sweetener in solutions and solid
form occurred above 80 and 140 °C, respectively.
The present study reports the interactions between steviol
glycosides and selected food antioxidants: vitamin C and
anthocyanins. These compounds can be found mainly in fruits
and fruit products, accounting for their health-promoting
properties and, in the case of anthocyanins, their sensory
quality. The impact on the stability of steviol glycosides during
storage was also determined, and the results were compared to
those obtained by other research teams. All of the tests were
performed on model solutions in conditions similar to those
prevailing in fruit juices and beverages.
■
MATERIALS AND METHODS
Materials. Highly purified steviol glycosides, rebaudioside A
(RebA, 96.2%) and stevioside (Ste, 98.5%), were obtained from
ChromaDex (Irvine, CA, USA). Certified standards of anthocyanins,
cyanidin-3-O-glucoside (C3G, 98.79%) and pelargonidin-3-O-glucoside (P3G, 99.22%), were purchased from Extrasynthese (Genay,
France). L-Ascorbic acid (99%) was obtained from POCh (Gliwice,
Poland). Acetonitrile for chromatography (HPLC grade) was acquired
from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were of
analytical grade and came from various suppliers.
Instrumentation. All chromatographic analyses were performed
using Waters (Milford, MA, USA) equipment: 2695 Separations
Module, 2995 Photodiode Array Detector, and 2414 Refractive Index
Detector. During preparation of the sample an ME235S analytical
balance (Sartorius AG, Göttingen, Germany) and an HI 211 pH meter
(Hanna Instruments, Woonsocket, RI, USA) were used.
Analytical Methods. Steviol glycosides were determined using a
method proposed by Bergs et al. with modifications.17 For the analysis
a Sunfire C8, 5 μm, 4.6 mm × 250 mm analytical column with a
Sunfire C8 Sentry guard cartridge, 5 μm, 4.6 mm × 20 mm (both
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t1/2 = ln 2/k1
din-3-glucoside (Table 1); the second series investigated interactions
between stevioside and ascorbic acid (Table 2). The component
cACN = concentration of ACN, cACN,0 = initial concentration of
ACN, k1 = rate of reaction, t = time, and t1/2 = half-life time.
The rates of this reaction were determined using linear
regression. The influence of the type of sweetener and its
concentration on the C3G and P3G half-life time is shown in
Figure 1. Detailed data can be found in Table S1 in the
Supporting Information.
Table 2. Composition of Model Solutions To Study the
Interactions between Steviol Glycosides and Vitamin Ca
symbol
AA
(mg L−1)
pH4.0/C
pH4.0/Ste200
pH4.0/Ste125
pH4.0/Ste50
pH4.0/Suc
pH3.6/C
pH3.6/Ste200
pH3.6/Ste125
pH3.6/Ste50
pH3.6/Suc
pH3.2/C
pH3.2/Ste200
pH3.2/Ste125
pH3.2/Ste50
pH3.2/Suc
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
Ste
(mg L−1)
Suc
(g L−1)
200
125
50
100
200
125
50
100
200
125
50
100
BA
(mg L−1)
buffer
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
4.0
4.0
4.0
4.0
4.0
3.6
3.6
3.6
3.6
3.6
3.2
3.2
3.2
3.2
3.2
(3)
a
The acidity of solutions has been set using citric acid−sodium
phosphate buffers prepared according to the method of Ruzin.20
Abbreviations: AA, ascorbic acid; BA, benzoic acid; Ste, stevioside;
Suc, sucrose.
Figure 1. Influence of sweetener type and concentration on
anthocyanin half-life times. Symbols and composition of model
solutions are described in Table 1. Error bars represent standard
deviation. Results were obtained from a duplicate analysis of two
independent samples.
concentrations and pH were set taking into account the values typical
for fruit juices and the legal requirements for steviol glycoside
concentration in beverages (applicable in the European Union).21
Samples were kept in the dark at ambient temperature for up to 60 and
7 days for the first and second series, respectively.
Statistical Analysis. Data were analyzed using Statistica 7.1
software (StatSoft, Tulsa, OK, USA). The statistical significance of the
differences in mean values was determined using an ANOVA test at α
= 0.05, and when significant differences existed, a Tukey test was
performed. The kinetic parameters of degradation reactions were
found using linear (C3G, P3G, AA) and nonlinear regression
(DHAA). All of the results were obtained from duplicate analyses of
two independent samples.
The addition of steviol glycosides, even at the highest
concentration of 200 mg L−1, did not affect the anthocyanin
degradation rate. Regardless of the glycoside used and its
concentration, the differences between the sample and control
were statistically insignificant. The addition of sugars at a
concentration of 100 g L−1 significantly increased the stability
of both anthocyanins by ca. 15% for a glucose−fructose mixture
and by ca. 33% for sucrose. The simultaneous use of both
sucrose and steviol glycosides turned out to be optimal with
respect to anthocyanin stabilization: the half-life values of C3G
and P3G were >50% higher than those observed in the control
samples. Pelargonidin-3-glucoside was more stable than
cyanidin-3-glucoside. This fact is consistent with the principle
that increased hydroxylation of the flavylium cation decreases
the stability of anthocyanins.28 The effect of sweeteners was
similar for both anthocyanins.
The influence of sugar on the stability of anthocyanin in
water solutions is dependent to a large extent on the
temperature, concentration, and type of sugar. Low concentrations, below 20%, of most carbohydrates stabilize pigments
during storage in cold and ambient temperatures; an exception
is fructose, which shows a destabilizing effect.29−31 The results
obtained in this study are consistent with these conclusions:
anthocyanins were more stable in solutions containing sugars,
and sucrose was also a better stabilizer than a mixture of
glucose and fructose. Steviol glycosides did not affect the
stability of anthocyanins, although they enhanced the protective
properties of carbohydrates. The mechanism of this process is
not yet known and should be examined in further research.
Kinetics of Vitamin C Degradation. The mechanism of
AA degradation is specific, as it depends on several factors,
whether it follows an aerobic or anaerobic pathway. The first
■
RESULTS AND DISCUSSION
Stability of Steviol Glycosides. Concentrations of
stevioside and rebaudioside in all of the model solutions
investigated in the study were measured on the first and last
days of each experiment (data not shown). The Tukey test did
not reveal statistically significant differences between the initial
and final concentrations of glycosides in any sample. These
results are consistent with previous studies. Chang and Cook11
found that steviol glycosides in carbonated drinks are stable at
room temperature. These results were later confirmed by many
authors examining various food matrices.9,10,12−16
Kinetics of Anthocyanin Degradation. Anthocyanins are
a group of flavylium cation based glycosides that act as
pigments and antioxidants in plant tissues. The main route of
their degradation leads through deglycosylation and cleavage to
aldehyde and benzoic acid derivatives.22,23 The kinetics of
anthocyanin (ACN) decrease is often described as a first-order
reaction with results expressed as half-life time values (eq
1−3).24−27
k1
ACN → product
(1)
cACN = cACN,0 exp( −k1t )
(2)
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step of both pathways leads to the formation of DHAA,
through ascorbic radical as intermediate in the anaerobic
pathway or the multistage aerobic pathway catalyzed by metal
ions. The second step is the irreversible hydrolysis to 2,3diketogulonic acid (DKGA). DHAA retains part of the
antioxidant and health-promoting activities of AA, although
DKGA and further metabolites do not have such properites.32
Reduction of DHAA to AA occurs in the presence of
reducing agents or enzymes such as dehydroascorbate
reductase. Lack of such compounds in model solutions allows
the above-mentioned reaction to be considered irreversible, so
the degradation of ascorbic acid can be regarded as two
consecutive first-order reactions (eq 4).33
k1
k2
AA → DHAA → DKGA
(4)
Changes in the concentration of compounds can be described
with differential equations. Their solutions (eqs 5 and 6) can be
used to determine the kinetic parameters of the reaction.
cAA = cAA,0 exp( −k1t )
c DHAA =
k1cAA,0
k 2 − k1
[exp(−k1t ) − exp(−k 2t )]
Figure 3. Influence of sweetener type, concentration, and acidity of
solution on ascorbic acid degradation rate. Symbols and composition
of model solutions are described in Table 2. Error bars represent
standard deviation. Results were obtained from a duplicate analysis of
two independent samples.
(5)
(6)
cAA = concentration of AA, cAA,0 = initial concentration of AA,
cDHAA = concentration of DHAA, k1 = rate of first reaction, k2 =
rate of second reaction, and t = time.
Rates of reaction were found using linear (AA) and nonlinear
regression (DHAA). The fit of the model kinetics to
experimental data is shown in Figure 2. The experimental
data are highly consistent with the assumed model of the
kinetics; for each sample the coefficient of determination was
>0.95 (significant at α = 0.05).
Figure 4. Influence of sweetener type, concentration, and acidity of
solution on dehydroascorbic acid degradation rate. Symbols and
composition of model solutions are described in Table 2. Error bars
represent standard deviation. Results were obtained from a duplicate
analysis of two independent samples.
effect was stronger: the rate of degradation was 26.6−41.9%
lower than in the control sample. Higher concentrations of
sweetener strengthened the protective effect; however, this
relationship was not linearly proportional.
The acidity of the solution influenced the stability of vitamin
C and the stabilizing effect of glycosides from S. rebaudiana.
Higher acidity of the solutions significantly improved the
protective effect of steviol glycosides on AA stability and, to a
lesser extent, on DHAA stability. In general, ascorbic acid
degradation is fastest in a pH close to 4.0 due to the maximum
occurrence of hydrogen ascorbate ions.32,34
Control samples containing sucrose (100 g L−1) were also
examined. The protective effect of this carbohydrate was
observed. Its strength did not depend on the acidity of the
solution.
The protective effect of steviol glycosides on the stability of
ascorbic acid was observed by Kroyer; however, his experiments
were very limited.9,10 Our research confirmed and expanded
these results: the protective effect applied to both vitamin C
forms, and it depended on the concentration of the sweetener
Figure 2. Fit between changes of ascorbic (AA) and dehydroascorbic
(DHAA) acid concentration computed using kinetic equations (eqs 5
and 6; lines) and experimental data (dots) in sample pH4.0/Ste200
(composition of sample in Table 2).
A graphical representation of the influence of the type of
sweetener and its concentration on the stability of vitamin C
(AA and DHAA) at various pH values is shown in Figure 3
(AA) and Figure 4 (DHAA). Detailed data can be found in
Table S2 in the Supporting Information.
The addition of steviol glycosides significantly increased the
stability of both ascorbic and dehydroascorbic acid. The rate of
degradation of ascorbic acid decreased by 4.0−22.3%, depending on the conditions. In the case of DHAA the protective
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and acidity of the sample. The protective effect of sucrose on
ascorbic acid is consistent with earlier publications.35,36
To our best knowledge this is the first study concerning the
influence of steviol glycosides on anthocyanin stability. It also
considerably expanded earlier experiments regarding interactions between stevioside and vitamin C.
The presented data demonstrate that the addition of
glycosides from S. rebaudiana can increase the stability of
vitamin C in water solutions of an acidic pH to a greater extent
than sucrose used in sensory equivalent amounts. The addition
of steviol glycosides also enhanced the protective effect of
sucrose on anthocyanins. This fact may be of practical
importance, indicating the additional benefits of using this
natural noncaloric sweetener for fruit beverages.
Our research was conducted on model solutions, which, on
the one hand, simplifies following the changes occurring in the
sample, but, on the other hand, eliminates the influence of
other constituents of real matrices, for example, radicals and
metal ions, which might significantly change the degradation
pattern through Fenton or Haber−Weiss reactions. The
observed phenomena should be further investigated in complex
food samples.37
■
ASSOCIATED CONTENT
S Supporting Information
*
Anthocyanin half-lives and ascorbic acid and dehydroascorbic
acid degradation rates. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*(L.W.) E-mail: [email protected]. Phone: (+48) 22
6063604. Fax: (+48) 22 8490426.
Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED
AA, ascorbic acid; ACN, anthocyanin (in general); BA, benzoic
acid; C3G, cyanidin-3-glucoside; DHAA, dehydroascorbic acid;
DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid;
HPLC, high-performance liquid chromatography; P3G, pelargonidin-3-glucoside; RebA, rebaudioside A; Ste, stevioside
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