Accepted Manuscript

Blackberry anthocyanins: β-cyclodextrin fortification for thermal and gastroin-

testinal stabilization

Ana fernandes, Marisa A.A. rocha, Luis M.N.B.F. Santos, Joana brás, Joana
oliveira, Nuno mateus, Victor de freitas

PII: S0308-8146(17)31747-8
DOI: https://doi.org/10.1016/j.foodchem.2017.10.109
Reference: FOCH 21932

To appear in: Food Chemistry

Received Date: 14 March 2017

Revised Date: 15 September 2017
Accepted Date: 19 October 2017

Please cite this article as: fernandes, A., rocha, M.A.A., Santos, L.M.N., brás, J., oliveira, J., mateus, N., de freitas,
V., Blackberry anthocyanins: β-cyclodextrin fortification for thermal and gastrointestinal stabilization, Food
Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.10.109

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
BLACKBERRY ANTHOCYANINS: β-CYCLODEXTRIN FORTIFICATION FOR THERMAL AND

GASTROINTESTINAL STABILIZATION

ANA FERNANDESa,*, MARISA A. A. ROCHAb, LUIS M. N. B. F. SANTOSb,

JOANA BRÁSa, JOANA OLIVEIRAa, NUNO MATEUSa & VICTOR DE FREITASa

a
REQUIMTE\LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do

Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.

b
CIQUP, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua

do Campo Alegre, s/n, 4169-007 Porto, Portugal.

*
Author to whom correspondence should be addressed, ana.fernandes@fc.up.pt

Tel: +351.220402597
ABSTRACT

Anthocyanins are potential food colorants due to their color, low toxicity and biological

properties. However, the low chemical stability of anthocyanins has limited their use.

In this work, the thermal stability of cyanidin-3-O-glucoside (cy3glc) (major blackberry

anthocyanin) and blackberry purees through molecular inclusion with β-cyclodextrin (β-CD)

was assessed. Complexation with β-CD showed a thermal stabilization of cy3glc, resulting on a

decrease of the degradation rate constant (k) and in several alterations in the cy3glc-β-CD DSC

thermogram. To assess the bioaccessibility of blackberry anthocyanins, the stability of

blackberry purees through simulated in vitro digestion was also studied. Despite the rapid

degradation of anthocyanins observed within the first minutes of simulated intestinal digestion,

complexation with β-CD allowed anthocyanins degradation to be slowed down. The results

obtained demonstrate the ability of β-CD to increase blackberry anthocyanins thermal stability

and also to decrease the rate of degradation of these pigments under simulated gastrointestinal

conditions.

Keywords: anthocyanins; cyclodextrin; DSC; encapsulation; gastrointestinal digestion; thermal

stability;
1. Introduction

Anthocyanins are vacuolar pigments widespread in Nature displaying a great diversity of

colors. These pigments can be incorporated in the food industry alternatively to the use of

synthetic dyes as they provide a high colorant capacity presenting also a low toxicity and water

solubility (Santos-Buelga, Mateus & De Freitas, 2014). Furthermore, anthocyanins may exert a

wide variety of biological activities, which raises the great interest in these compounds as

colorants in the Food Industry (Clifford, 2000; He & Giusti, 2010). However, the application of

anthocyanins as food colorants has proved to be a major technological challenge since these

compounds have a low chemical stability. The common anthocyanins show instability toward a

variety of chemical and physical parameters including pH values, oxygen, high temperatures

and light (Andersen & Jordheim, 2013). Anthocyanins can also be involved in chemical and

enzymatic reactions which may degrade them to colorless products or transform them into new

structures (He et al., 2012). Besides this, their health promoting effects are also hindered by

their low stability under the physicochemical conditions they are exposed to after oral

consumption by humans affecting their bioaccessibility and further bioavailability (Fleschhut,

Kratzer, Rechkemmer & Kulling, 2006; Flores et al., 2015). In fact, the search for improved

processing methods to better control anthocyanin losses and/or to address anthocyanin reactions

in the right direction to obtain more stable and desirable colors constitute a major challenge for

the food industry (Santos-Buelga, Mateus & De Freitas, 2014).

Interaction between polysaccharides and anthocyanins has also shown to be especially

important in the understanding of anthocyanins stabilizing mechanisms (Buchweitz, Speth,

Kammerer & Carle, 2013; Wu, Guan & Zhong, 2015). In this context, molecular inclusion may

provide a stabilizing environment for anthocyanins with cyclodextrins being a potential

candidate for pigment inclusion (Flores et al., 2015; Howard, Brownmiller, Prior &

Mauromoustakos, 2013; Mourtzinos et al., 2008). β-CD is a cyclic oligosaccharide with seven

glucose residues linked by α-(1-4) glycosidic bonds in a truncated cone-shaped structure. The
most important functional property of β-CD is its ability to form non-covalent inclusion

complexes with a wide range of guest molecules due to the adaptable hydrophobic

tridimensional cavity. The inclusion complex of these host–guest systems occurs through

various interactions such as hydrogen bonding, van der Waals interaction and also electrostatic

attraction (Le Bourvellec, 2003). The inclusion complexes can affect some physicochemical

properties of anthocyanins, particularly concerning water solubility, solution stability and even

equilibrium and rate constants (Basilio et al., 2013). Previous works have also shown an

improved thermal stability of anthocyanin-rich extracts through the molecular inclusion with β-

CD, a fundamental feature to be considered for the putative technological applications of

anthocyanins (Mourtzinos et al., 2008).

On the other hand, interaction between anthocyanins and β-CD are likely to affect

anthocyanins bioavailability, potentially providing an important mean to increase concentrations

of bioactive anthocyanins in the gastric tract. This could potentially affect the nutritional content

and the functional potential of anthocyanin-rich products and thus favor their beneficial health

effects (Cheynier, 2005; Oidtmann et al., 2012; Padayachee et al., 2012). β-cyclodextrins have

the capacity to protect bioactive food components from the deleterious conditions in the

stomach and upper small bowel, allowing them to be liberated in the colon (Kosaraju, 2005) and

thus possibly boost their beneficial effects (Flores et al., 2015).

Therefore, improving the thermal stability at weakly acidic conditions (typical pH of food

products that contain anthocyanins) while boosting the gastrointestinal stability of anthocyanins

could reinforce anthocyanins applications as food colorants. The aims of this work were to

investigate the effect of the molecular inclusion of cyanidin-3-O-glucoside with β-CD,

comparing the thermal stability between nonencapsulated and encapsulated anthocyanins. It was

also intended to evaluate the gastrointestinal stability of anthocyanins in blackberry purees

using an in vitro digestion model.

2. Materials and methods

2.1. Materials

The chemicals used were all analytical or HPLC grade. General chemicals and reagents were

obtained from Sigma-Aldrich® (Madrid, Spain). Cy3glc was extracted and purified from

blackberries (Rubus fruticosus) by semipreparative HPLC with C18 reversed phase column (250

mm x 4.6 mm i.d., 5 µm, Merck, Lichrospher), as described elsewhere (Azevedo et al., 2010).

Blackberries were obtained from Verdes Quimeras Ltd.

2.2. Thermal degradation studies

To study anthocyanins thermal stability, a stock solution of cy3glc (0.2 mM) was prepared in

water and left under stirring for 24 hours. The molecular inclusion complexes were prepared by

dissolving cy3glc in a β-CD solution at an approximate ratio of 1:10 (w:w, cy3glc:β-CD) and

was also left under stirring for 24 hours. The pH of each solution was adjusted at pH 4 with HCl

(0.1 M). All systems were subsequently frozen at -20 ºC and then freeze-dried. Then, the

thermal stability of cy3glc and cy3glc-β-CD complexes was studied in aqueous solution (pH 4,

which is typical of food products that contain anthocyanins) (Mourtzinos et al., 2008). Briefly,

aliquots of 10 mL of cy3glc solution (0.2 mM) and cy3glc-β-CD solution (at the same

anthocyanin concentration) were placed in hydrolysis tubes in a thermostatic oil bath and heated

at 60 and 90º C for 120 minutes. At predetermined intervals, aliquots of each sample were

removed from the oil bath and rapidly cooled at -20º C. Before analysis, the temperature of each

sample was allowed to reach room temperature and total anthocyanin content was determined

by HPLC-DAD.

The thermal stability of fortified (1:10; w:w; cy3glc:β-CD) and non-fortified blackberry

purees (15g frozen blackberries/60 mL water) was also studied. This study was performed as
previously described and total anthocyanin content was determined by HPLC-DAD in the

supernatants after centrifugation (20 minutes at 13000 rpm).

2.3. HPLC-DAD and LC-DAD/ESI-MS analysis

Anthocyanins were analyzed and quantified using a HPLC-DAD system (Merck Hitachi)

consisting on an Elite Lachrom L-2130 pump, with a reversed-phase C18 column (250 mm x 4.6

mm i.d., 5 µm, Merck, Lichrospher), thermostatized at 25 ºC. Detection was carried out at 520

nm on an Elite Lachrom L-2455 Diode Array Detector. 20 µL of each sample was injected

using an Elite Lachrom L-2200 autosampler and anthocyanins were separated and quantified

using a mobile phase, a flow rate and an elution gradient as described elsewhere (Oliveira et al.,

2013).

The identification of anthocyanins present in the blackberry purees was performed by LC-

DAD/ESI-MS analysis, using a Finnigan Surveyor series liquid chromatograph, equipped with a

150x4.6 mm i.d., 5 µm, (Merck, Lichrospher) reversed-phase C18 column, thermostatized at 25

ºC. Detection was carried out between 200 and 700 nm using a Finnigan Surveyor PDA Plus

detector. The mass detection was carried out by a Finnigan LCQ DECA XP MAX (Finnigan

Corp., San José, Calif., USA) mass detector with an API (Atmospheric Pressure Ionisation)

source of ionisation and an ESI (ElectroSpray Ionisation) interface. Solvents were A:

H2O/HCOOH (99:1) and B: HCOOH/H2O/CH3CN (1:69:30) and the LC gradient used was the

same as described elsewhere (Oliveira et al., 2013), except for the flow rate which was 0.5

ml/min. The capillary voltage was 4 V and the capillary temperature 325 ºC. Spectra were

recorded in positive ion mode between m/z 0 and 2000. The mass spectrometer was

programmed to do a series of three scans: a full mass, a zoom scan of the most intense ion in the

first scan, and a MS–MS of the most intense ion using relative collision energy of 30 and 60 V.

2.4. Evaluation of thermal degradation kinetics

 The data obtained by HPLC-DAD analysis (anthocyanin content, n=3 trials) was fitted by a

first-order kinetic model (equation (1)), where C0 is the initial anthocyanin content and C is the

anthocyanin content after predetermined time (t) (Wang & Xu, 2007). Degradation rate

constants (k) were obtained from the slope of a plot of the natural logarithmic of anthocyanin

retention (ln(C/C0) versus time (t). For a first order reaction, half-life values (t1/2, the time

needed for 50% degradation of anthocyanins) were determined by equation (2) (Ozkan,

Yemenicioglu, Asefi & Cemeroglu, 2002).

ln (C/C0) = -kt (1)

t½= ln (2)/ k (2)

2.5. Differential scanning calorimetry (DSC)

The thermal behavior and thermal stability of β-CD, cy3glc and its β-CD complex was

evaluated by differential scanning calorimetry (DSC). DSC measurements were performed

using a power compensation type differential scanning calorimeter, model SETARAM DSC

141, using a heating rate of 10 ºC min–1 and hermetically sealed 50 µL aluminum crucibles. The

experiments were done under constant flow of nitrogen, 50 cm3 min–1. The temperature and heat

flux scales were calibrated by measuring the temperature and the enthalpy of fusion of some

reference materials (Sabbah et al., 1999): o-terphenyl, benzoic acid, indium, triphenylene, tin,

perylene and zinc. Approximately 2 mg of cy3glc, β-CD or a quantity of the complex cy3glc-β-

CD containing the same amount of anthocyanin were placed in aluminum crucibles and were

heated from room temperature to 25 ºC in nitrogen atmosphere at a heating rate of 10 ºC min–1 .

The samples remained at 25 ºC for 10 min in order to ensure a homogeneous temperature

distribution within the sample and then were heated up to 400 ºC at the same heating rate.

2.6. In vitro digestion model

To study the possible impact of β-CD on the bioacessibility of food anthocyanins, simulated

in vitro gastric and small intestine digestion was carried out according the procedure described

by Netzel, 2011 and Padayachee, 2013 (Netzel et al., 2011; Padayachee et al., 2013). Briefly,
blackberry purees (15 g frozen blackberries/60 mL water) was equally divided into 15 mL

screw-cap vials. One of the samples was fortified with β-CD (1:10; w:w; cy3glc:β-CD), the pH

of both samples was adjusted to pH 4.0 and both of them were left under agitation for 18 hours.

Both samples were subjected to simulated gastric digestion followed by intestinal digestion.

Before the gastric digestion, the pH of the reconstituted puree samples was adjusted to pH 2.0

with the addition of HCl (6 M) to simulate the lowest pH of the gastric environment. To these

samples, 1000 µL pepsin solution (10 mg/mL pepsin from porcine gastric mucosa dissolved in

HCl (0.1 M)) was added and the mixtures were incubated for 1 h in a shaking bath at 37 ºC.

Three aliquots (500 µL) were taken at 0, 30 and 60 minutes of incubation. After 60 minutes of

simulated gastric digestion and to inactivate pepsin, the pH of the samples was gradually

increased up to pH 5.7 by adding NaHCO3 in 12 Mm CaCL2 and the samples were further

incubated for 30 minutes in a 37 ºC shaking bath. To initiate the intestinal digestion, the pH of

the mixture was increased to 7.0 by adding NaOH (1 M) followed by the addition of 1 mL of

pancreatin-bile solution (1 mg/mL pancreatin from porcine pancreas and 12 mg/mL porcine bile

extract in NaHCO3 (0.1 M)). The mixtures were further incubated in a shaking bath at 37 ºC for

120 min and aliquots were taken at 0, 15, 30, 60, 90, and 120 minutes of intestinal digestion.

Samples (500 µL) from various digestion time points were stored frozen at −80 °C until further

HPLC-DAD analysis. Total anthocyanin content in blackberry purees was measured in the

supernatants after centrifugation (20 minutes at 13,000 rpm).

2.7. Statistical analysis

Statistical analysis was performed using GraphPad Prism statistical software. Statistical

significance values were made by one-way analysis of variance with Tukey post hoc test or by

two-tailed t test. The statistical analysis performed were considered significant when p<0.0001.

3. Results and discussion

3.1. Thermal degradation studies

 In general, linear relationships were observed between ln (C/C0 ) and time for both control

(cy3glc) and complex sample (cy3glc-β-CD) (R2 between 0.9552 and 0.9926), apparently

indicating a first order reaction kinetics for anthocyanin degradation. Similar kinetic behaviors

for anthocyanins have already been reported by other authors (Ersus & Yurdagel, 2007;

Mourtzinos et al., 2008). The first order degradation rate constant (k) obtained from the slope of

logarithmic plots of cy3glc retention versus time and the half-life value (t1/2, the time needed for

50% degradation of anthocyanins) calculated from the degradation rates are shown in Table 1.

As expected, the degradation rate constant of cy3glc increased with increasing heat temperature

and with time. The results show that the effect of temperature on the degradation kinetic rates

for cy3glc in control and complexed with β-CD were significantly different (p<0.05) for both

temperatures tested. Comparison of k and t1/2 values revealed that cy3glc was more stable in the

presence of β-CD. This possible anthocyanin stabilization could be due to anthocyanin

complexation with β-CD. The equilibrium forms of anthocyanins present at pH 4.0 are the

flavylium cation, the hydrated hemiketal (major specie ≈ 75%), the chalcone forms and the

quinoidal base form (Leydet et al., 2012). As pointed out in a previous work, (Fernandes et al.,

2014a; Fernandes et al., 2013) a preferential inclusion of the neutral and more flexible hemiketal

forms is expected. At pH 3.5 anthocyanin thermal degradation is generally accepted to be

initiated by the hydrolytic opening of the pyrylium ring to form the chalcone-glycoside form

(Sadilova, Stintzing & Carle, 2006). The chalcone-glycoside would then be cleaved to yield the

corresponding chalcones, which degrades to the hydroxybenzaldehyde and hydroxybenzoic acid

derivatives. The significant protection of cy3glc towards thermal degradation observed in the

presence of β-CD could be a result of the preferential inclusion of the hydrated forms that could

prevent extended tautomerization reaction leading to the formation of the chalcone form and

subsequent formation of hydroxybenzaldehyde and 4-hydroxybenzoic acid anthocyanin

derivatives.
Table 1. Degradation kinetics parameters of cy3glc and cy3glc-β-CD complex (n=3 trials).
Statistical significance values were made by one-way analysis of variance with Tukey post hoc
test. Columns with the same symbol (*, **) indicate that there is significance difference
(p<0.0001).

Temperature k x103(min-1) t1/2 (h)

Cy3glc-
Cy3glc* Cy3glc** β CD**
Cy3glc-β
β CD*

60º C -0.8 -0.29 14 ± 3 41 ± 5

± 0.2 ± 0.03
90º C -3.2 -2.6 3.6 ± 0.1 4.4 ± 0.2
± 0. 1 ± 0.1

3.2. Study of the decomposition of cyanidin-3-O-glucoside and its β-CD inclusion complex by

DSC

DSC is widely used to confirm the formation of a complex in the solid state. For instance,

the disappearance of thermal events of guest molecules, such as endothermic peak assigned to

melting, when examined as cyclodextrins inclusion complexes is generally considered as a

proof of inclusion (Pralhad & Rajendrakumar, 2004). Moreover, DSC can be used to study the

thermal degradation of a compound. Fig. 1 shows the combined DSC thermograms of cy3glc

(blue line), β-CD (green line) and of the inclusion complex of cy3glc-β-CD (red line) as a

function of temperature. Through the thermogram analysis there was an indication of complex

formation, between the anthocyanin and β-CD, based on the shape of the dehydration process in

the complex. In the complex form, cy3gc dehydration process was shifted to higher

temperatures. It was also noticed a decrease on the thermal stability of the dehydration process

in the β-CD in the complex form (a shift of 293.15 K) (A). The DSC thermogram of cy3glc
(blue line) demonstrated the occurrence of a decomposition or polymerization process at 170º C

(B). However, after complexation with β-CD this decomposition process is not present in the

thermogram of the complex (red line), which is a good indication of anthocyanin-β-CD

interaction. The absence of this peak in the thermogram of the complex could also be an

indication of the anthocyanin thermal protection due to inclusion formation. In the complex

form, the pre-melting peak was shifted to higher temperature (a shift of 303.15 K) than the

previous observed temperature of melting for cy3glc and pre-melting for β-CD (C). This

indicates a significant interaction between cy3glc and β-CD that is maintained until the pre-

melting temperature of β-CD. The shifted to higher temperature is followed and supported by an

increase of the process enthalpy.

Fig. 1. DSC thermograms of cy3glc (blue line), β-CD (green line) and inclusion complex
(cy3glc-β-CD) (red line) under nitrogen atmosphere.

Even though molecular inclusion of pure cy3glc has proven to be an efficient method to

increase anthocyanins thermal stability, it was important to verify the impact of β-CD

fortification on the thermal behavior of anthocyanins in a food matrix. For this reason,

blackberry puree was chosen as a model food product to study anthocyanins thermal stability.

Prior to this study, the anthocyanin content in blackberry purees was determined. The total

anthocyanin content in blackberry purees was quantified after centrifugation (20 min at 13000

rpm) by HPLC-DAD and characterized by LC-DAD/ESI-MS. A typical HPLC chromatogram

of blackberry purees is presented in Fig. 2. Four main peaks could be identified by LC-MS

corresponding to cyanidin derivatives. Cyanidin-3-O-glucoside was the major anthocyanin

present in the purees accounting for approximately 89% of total anthocyanins concentration,

complemented by other three minor compounds such as cyanidin-3-O-dioxalyl-glucoside (6%),

cyanidin-3-O-xylose (3%) and cyanidin-3-O-(6’’-malonyl-glucoside) (2%). For this reason, all

calculations were based on the molecular weight of cyanidin-3-O-glucoside. The total

anthocyanin concentration of blackberry puree was 0.4 mg/mL.

Fig. 2. HPLC chromatogram of anthocyanins (520 nm) from blackberries purees. Peak
assignment based on literature report (Fan-Chiang & Wrolstad, 2005; Marques et al., 2016): 1:
Cyanidin-3-O-glucoside (cy3glc), 2: Cyanidin-3-O-xylose (cy3xyl), 3: Cyanidin-3-O-(6’’-
malonyl-glucoside) (cy3malglc), 4: Cyanidin-3-O-dioxalyl-glucoside (cy3dioxaglc).

3.3. Thermal stability of blackberries purees

The kinetic plots of fortified and non-fortified blackberry purees at 90 ºC are shown in Fig.

3. As for pure cy3glc, linear relationships were observed between ln (C/C0) and time for both

non-fortified and β-CD fortified purees (R2 between 0.9911 and 0.9942, respectively), also

indicating a first order reaction kinetics for anthocyanin degradation for both situations. The

results showed that the effect of temperature on the degradation kinetic rates for non-fortified

purees and β-CD fortified were different with the rate of degradation being lower in the

presence of β-CD. Comparison of t1/2 values also revealed that anthocyanins were more resistant

in the presence of β-CD as the presence of this macrocycle increased the half-time value. At

90ºC and after 120 min, the puree fortified with β-CD retained approximately 71.6% (±0.4) of
total anthocyanins, compared to 65.8% (±0.6) retention for the corresponding puree with no β-

CD addition. The protective effect of β-CD was evident for all blackberry anthocyanins and was

consistent with previous studies in which addition of β-CD to chokeberry juices improved

anthocyanin retention (Howard, Brownmiller, Prior & Mauromoustakos, 2013). When

compared to the data obtained for pure cy3glc, it was observed a lower β-CD effect on

anthocyanin thermal stabilization in the blackberry puree. This could be due to the presence of

other blackberries compounds (e.g. other polyphenolic compounds) that could preferentially

interact with β-CD, causing a lower anthocyanin protective effect. However, to clarify the

effects of other food matrix components on anthocyanin-β-CD interactions a set of different

temperatures should be tested.

Fig. 3. Degradation kinetics of non-fortified (•) and β-CD fortified (▪) blackberries puree at 90º
C. Data is presented as mean ± SD of n=3 trials (A). Degradation kinetics parameters of non-
fortified and β-CD fortified blackberries puree (k and t1/2) are also shown. Statistical
significance values were made by two-tailed t test. Columns with the same symbol (*, **)
indicate that there is significance difference (p<0.0001).

Although interaction between cy3glc and blackberry anthocyanins with β-CD has shown to

be a promising method to increase the thermal stability of anthocyanins, a fundamental issue for

the technological application in the Food Industry, it was also important to assess the impact of

this molecular interaction on the gastrointestinal stability. In fact, β-CD could protect

anthocyanins from the deleterious gastrointestinal conditions resulting in an improved

bioavailability, which could also reinforce anthocyanins applications as food colorants.

 3.4. Anthocyanins stability under simulated gastrointestinal digestion

The stability of blackberry puree (β-CD fortified vs non-fortified blackberries purees) was

subsequently assessed using an in vitro digestion model that simulated the physiochemical

changes in the upper gastrointestinal tract. Nevertheless, such simulated medium must always

be recognized as an approximation of in vivo conditions, which vary widely in several aspects

like pH, concentration of bile salts and phospholipids and mineral composition (Oidtmann et al.,

2012).

In previous works, the structure of cy3glc-β-cyclodextrin complexes and the impact of these

complexes on the color and on the network of chemical reactions was studied. It was observed

that β-cyclodextrin promoted discoloration of anthocyanins, the so-called anti-copigmentation

effect (Dangles, Stoeckel, Wigand & Brouillard, 1992), affecting mainly the hydration reaction

(Fernandes et al., 2013). These results suggested the preferable interaction between β-CD and

anthocyanins hemiketal form and, as anticipated, during the current experiments a decrease on

the color of blackberry puree was observed in the presence of β-CD, corresponding to the anti-

copigmentation effect (data not shown). Addition of β-CD induced a decrease of approximately

38% of the anthocyanins visible absorption band.

During simulated gastric and intestinal digestion, samples at various time points were

centrifuged with the supernatant being removed and analyzed by HPLC-DAD. The initial

anthocyanin concentration was approximately 0,4 mg/mL and the concentrations in all samples

were related to this value. At preset time points (0, 30 and 60 min for gastric digestion; 0, 30,

60, 90 and 120 min for intestinal digestion) 500 µL of each sample were collected. The

intestinal digestion samples were acidified with HCl (6M) to stabilize anthocyanins. The

degradation kinetics of non-fortified and β-CD fortified blackberry anthocyanins under gastric

and intestinal simulated conditions are shown in Fig. 4. The results presented correspond to total

anthocyanin concentration (four anthocyanins present in blackberries) although the behavior

observed only for cy3glc (major blackberry anthocyanin) was the same.
Fig. 4. Anthocyanin Degradation Index (ADI) during the mimicked gastric and intestinal
digestion of blackberry puree and β-CD fortified blackberry puree. Data (means ± SD of n=3
trials) is presented as percent of anthocyanin degradation (degraded amounts vs. initial
anthocyanin concentration). Statistical significance values were made by two-tailed t test. Data
with the same symbol (*) indicate that there is significance difference (p<0.002).

In the stomach mimetic medium the total anthocyanin concentration did not decrease

significantly during the incubation time and its concentration remained roughly constant for 60

min in accordance with preferable stability of anthocyanins at low pH (Clifford, 2000). Total

anthocyanin concentration only started to decrease by the end of the incubation (60 minutes).

Likewise, the stability of anthocyanins in β-CD fortified purees was similar to the non-fortified

purees with the amounts of anthocyanins degraded at the end of gastric digestion being very

similar. These results are consistent with the indication that anthocyanins are not degraded

under stomach conditions (Oidtmann et al., 2012). Although anthocyanins were stable at lower

pH values (gastric environment) they were rapidly degraded at higher pH values. After 60

minutes of simulated gastric digestion and to inactivate pepsin, the pH of the samples was

gradually increased up to pH 5.7 and further incubated for 30 min. Subsequently, and to initiate

the intestinal digestion, the pH of the mixture was additionally increased up to 7.0. After pH

increase up to ≈ pH 6 and in the beginning of the second phase of this in vitro digestion process,

a large decrease on anthocyanin concentration was observed for both the β-CD fortified and
non-fortified blackberry purees. These results seemed to indicate that the intestinal digestion

phase with much higher pH is of major importance in the irreversible breakdown of the

anthocyanins. When reaching intestine, pH shifts with anthocyanins being converted into

unstable quinoidal base (Fernandes et al., 2014b; Woodward, Kroon, Cassidy & Kay, 2009).

Prolonged exposure may have enhanced degradation between the B and C rings resulting in the

destruction of the anthocyanin chromophore (Clifford, 2000; McDougall, Fyffe, Dobson &

Stewart, 2005). Particularly, within 15 min post initiation of the intestinal digestion 71% (±4%)

of anthocyanins from blackberry puree were degraded. Compared with free anthocyanin

solution, the rate of anthocyanin degradation in the presence of β-CD was slightly lower over

the time course. Anthocyanin-β-CD complexes showed a better stability in simulated intestinal

digestion (55±4% of anthocyanins degradation for β-CD fortified puree). β-CD fortification and

the possible occurrence of molecular inclusion seemed to have provided a more stabilizing

environment to protect anthocyanins thus diminishing pigment degradation. At 120 min of

intestinal digestion, approximately 37 and 44% of total anthocyanins were found in the

supernatants from non-fortified and fortified blackberry purees, respectively.

4. Conclusions

This study demonstrated that anthocyanins were protected from thermal degradation by the

presence of β-CD, which could be an advantage for efficient utilization in food systems.

Moreover, the in vitro stability of anthocyanins during simulated gastrointestinal digestion was

slightly improved in β-CD fortified blackberry puree, with this interaction resulting in lower

pigment degradation. Therefore, anthocyanin-loaded β-CD could potentially carry and stabilize

anthocyanins improving their bioavailability. From a technological perspective, the results

obtained in this study contribute to the increasing knowledge regarding the phenomena that

drive the stabilization and the improvement of anthocyanins bioavailability and bio-

effectiveness in vivo.
Acknowledgments

This work was financially supported by FEDER national funds through COMPETE, POPH/FSE

and QREN through projects UID/QUI/50006/2013 and PTDC/AGR-TEC/2789/2014

and by FCT (Fundação para a Ciência e Tecnologia) to CIQUP, University of Porto (strategic

project PEst- C/QUI/UI0081/2011) and through one Post-PhD grant

(SFRH/BPD/112465/2015) and one IF starting grant IF/00225/2015. To all financing

sources the authors are greatly indebted.

References

Andersen, O., & Jordheim, M. (2013). Basic anthocyanin chemistry and dietary sources. In: T. C.
Wallace, & M. Giusti, Anthocyanins in Health and Disease (pp. 13-90). Boca Raton, Florida,
USA: CRC Press.
Azevedo, J., Fernandes, I., Faria, A., Oliveira, J., Fernandes, A., de Freitas, V., & Mateus, N.
(2010). Antioxidant properties of anthocyanidins, anthocyanidin-3-glucosides and respective
portisins. Food Chemistry, 119(2), 518-523.
Basilio, N., Fernandes, A., de Freitas, V., Gago, S., & Pina, F. (2013). Effect of β-cyclodextrin on
the chemistry of 3',4',7-trihydroxyflavylium. New Journal of Chemistry, 37(10), 3166-3173.
Buchweitz, M., Speth, M., Kammerer, D. R., & Carle, R. (2013). Impact of pectin type on the
storage stability of black currant (Ribes nigrum L.) anthocyanins in pectic model solutions. Food
Chemistry, 139(1–4), 1168-1178.
Cheynier, V. (2005). Polyphenols in foods are more complex than often thought. The American
Journal of Clinical Nutrition, 81(1), 223S-229S.
Clifford, M. N. (2000). Anthocyanins - nature, occurrence and dietary burden. Journal of the
Science of Food and Agriculture, 80(7), 1063-1072.
Dangles, O., Stoeckel, C., Wigand, M. C., & Brouillard, R. (1992). Two very distinct types of
anthocyanin complexation: Copigmentation and inclusion. Tetrahedron Letters, 33(36), 5227-
5230.
Ersus, S., & Yurdagel, U. (2007). Microencapsulation of anthocyanin pigments of black carrot
(Daucuscarota L.) by spray drier. Journal of Food Engineering, 80(3), 805-812.
Fan-Chiang, H.-J., & Wrolstad, R. E. (2005). Anthocyanin Pigment Composition of Blackberries.
Journal of Food Science, 70(3), C198-C202.
Fernandes, A., Ivanova, G., Brás, N. F., Mateus, N., Ramos, M. J., Rangel, M., & de Freitas, V.
(2014a). Structural characterization of inclusion complexes between cyanidin-3-O-glucoside
and β-cyclodextrin. Carbohydrate Polymers, 102(0), 269-277.
Fernandes, A., Sousa, A., Azevedo, J., Mateus, N., & de Freitas, V. (2013). Effect of cyclodextrins
on the thermodynamic and kinetic properties of cyanidin-3-O-glucoside. Food Research
International, 51(2), 748-755.
Fernandes, I., Faria, A., Calhau, C., de Freitas, V., & Mateus, N. (2014b). Bioavailability of
anthocyanins and derivatives. Journal of Functional Foods, 7, 54-66.
Fleschhut, J., Kratzer, F., Rechkemmer, G., & Kulling, S. E. (2006). Stability and
biotransformation of various dietary anthocyanins in vitro. European Journal of Nutrition,
45(1), 7-18.
Flores, G., Ruiz del Castillo, M. L., Costabile, A., Klee, A., Bigetti Guergoletto, K., & Gibson, G. R.
(2015). In vitro fermentation of anthocyanins encapsulated with cyclodextrins: Release,
metabolism and influence on gut microbiota growth. Journal of Functional Foods, 16, 50-57.
He, F., Liang, N. N., Mu, L., Pan, Q. H., Wang, J., Reeves, M. J., & Duan, C. Q. (2012).
Anthocyanins and Their Variation in Red Wines I. Monomeric Anthocyanins and Their Color
Expression. Molecules, 17(2), 1571.
He, J. A., & Giusti, M. M. (2010). Anthocyanins: Natural Colorants with Health-Promoting
Properties. In: M. P. Doyle, & T. R. Klaenhammer, Annual Review of Food Science and
Technology, Vol 1, vol. 1 (pp. 163-187). Palo Alto: Annual Reviews.
Howard, L. R., Brownmiller, C., Prior, R. L., & Mauromoustakos, A. (2013). Improved Stability of
Chokeberry Juice Anthocyanins by β-Cyclodextrin Addition and Refrigeration. Journal of
Agricultural and Food Chemistry, 61(3), 693-699.
Kosaraju, S. L. (2005). Colon Targeted Delivery Systems: Review of Polysaccharides for
Encapsulation and Delivery. Critical Reviews in Food Science and Nutrition, 45(4), 251-258.
Le Bourvellec, C. (2003). Association entre les procyanidols et les polymères pariétaux de
pommes: quantification et conséquences. vol. Docteur: Universite de Rennes I.
Leydet, Y., Gavara, R., Petrov, V., Diniz, A. M., Parola, A., Lima, J. C., & F., P. (2012). The effect
of self-aggregation on the determination of the kinetic and thermodynamic constants of the
network of chemical reactions in 3-glucoside anthocyanins. Phytochemistry, 83(0), 125-135.
Marques, C., Fernandes, I., Norberto, S., Sá, C., Teixeira, D., de Freitas, V., Mateus, N., Calhau,
C., & Faria, A. (2016). Pharmacokinetics of blackberry anthocyanins consumed with or without
ethanol: A randomized and crossover trial. Molecular Nutrition & Food Research, n/a-n/a.
McDougall, G. J., Fyffe, S., Dobson, P., & Stewart, D. (2005). Anthocyanins from red wine –
Their stability under simulated gastrointestinal digestion. Phytochemistry, 66(21), 2540-2548.
Mourtzinos, I., Makris, D. P., Yannakopoulou, K., Kalogeropoulos, N., Michali, I., & Karathanos,
V. T. (2008). Thermal Stability of Anthocyanin Extract of Hibiscus sabdariffa L. in the Presence
of β-Cyclodextrin. Journal of Agricultural and Food Chemistry, 56(21), 10303-10310.
Netzel, M., Netzel, G., Zabaras, D., Lundin, L., Day, L., Addepalli, R., Osborne, S. A., & Seymour,
R. (2011). Release and absorption of carotenes from processed carrots (Daucus carota) using in
vitro digestion coupled with a Caco-2 cell trans-well culture model. Food Research
International, 44(4), 868-874.
Oidtmann, J., Schantz, M., Mäder, K., Baum, M., Berg, S., Betz, M., Kulozik, U., Leick, S.,
Rehage, H., Schwarz, K., & Richling, E. (2012). Preparation and Comparative Release
Characteristics of Three Anthocyanin Encapsulation Systems. Journal of Agricultural and Food
Chemistry, 60(3), 844-851.
Oliveira, J., da Silva, M. A., Jorge Parola, A., Mateus, N., Brás, N. F., Ramos, M. J., & de Freitas,
V. (2013). Structural characterization of a A-type linked trimeric anthocyanin derived pigment
occurring in a young Port wine. Food Chemistry, 141(3), 1987-1996.
Ozkan, M., Yemenicioglu, A., Asefi, N., & Cemeroglu, B. (2002). Degradation kinetics of
anthocyanins from sour cherry, pomegranaye, and strawberry juices by hydrogen peroxide.
Journal of Food Science, 67, 525-529.
Padayachee, A., Netzel, G., Netzel, M., Day, L., Mikkelsen, D., & Gidley, M. J. (2013). Lack of
release of bound anthocyanins and phenolic acids from carrot plant cell walls and model
composites during simulated gastric and small intestinal digestion. Food & Function, 4(6), 906-
916.
Padayachee, A., Netzel, G., Netzel, M., Day, L., Zabaras, D., Mikkelsen, D., & Gidley, M. J.
(2012). Binding of polyphenols to plant cell wall analogues – Part 1: Anthocyanins. Food
Chemistry, 134(1), 155-161.
Pralhad, T., & Rajendrakumar, K. (2004). Study of freeze-dried quercetin-cyclodextrin binary
systems by DSC, FT-IR, X-ray diffraction and SEM analysis. Journal of Pharmaceutical and
Biomedical Analysis, 34, 333-339.
Sabbah, R., Xu-wu, A., Chickos, J. S., Leitão, M. L., Roux, M. V., & Torres, L. A. (1999). Reference
materials for calorimetry and differential thermal analysis. Thermochimica Acta 331, 93–204.
Sadilova, E., Stintzing, F. C., & Carle, R. (2006). Thermal Degradation of Acylated and
Nonacylated Anthocyanins. Journal of Food Science, 71(8), C504-C512.
Santos-Buelga, C., Mateus, N., & De Freitas, V. (2014). Anthocyanins. Plant Pigments and
Beyond. Journal of Agricultural and Food Chemistry, 62(29), 6879-6884.
Wang, W.-D., & Xu, S.-Y. (2007). Degradation kinetics of anthocyanins in blackberry juice and
concentrate. Journal of Food Engineering, 82(3), 271-275.
Woodward, G., Kroon, P., Cassidy, A., & Kay, C. (2009). Anthocyanin Stability and Recovery:
Implications for the Analysis of Clinical and Experimental Samples. Journal of Agricultural and
Food Chemistry, 57(12), 5271-5278.
Wu, J., Guan, Y., & Zhong, Q. (2015). Yeast mannoproteins improve thermal stability of
anthocyanins at pH 7.0. Food Chemistry, 172, 121-128.
HIGHLIGHTS

• Anthocyanins are potential food colorants;

• Thermal stability of cyanidin-3-O-glucoside and blackberry purees were
enhanced in the presence of β-cyclodextrin;
• β-cyclodextrin encapsulation decreased anthocyanins rate of degradation under
simulated gastrointestinal conditions;