G.A. Cardoso-Ugarte

LWT - Food Science and Technology 59 (2014) 276e282
Contents lists available at ScienceDirect
LWT - Food Science and Technology
journal homepage: www.elsevier.com/locate/lwt
Microwave-assisted extraction of betalains from red beet (Beta
vulgaris)
G.A. Cardoso-Ugarte a, M.E. Sosa-Morales b, T. Ballard d, A. Liceaga c,
lez c, *
M.F. San Martín-Gonza
a
rtir S/N, San Andr
Departamento de Ingeniería en Alimentos, Universidad de las Am
ericas-Puebla, Ex-Hacienda Santa Catarina Ma
es Cholula CP 7281,
Puebla, Mexico
b
n Ciencias de la Vida, Campus Irapuato-Salamanca, Universidad de Guanajuato, Carr. Irapuato-Silao km 9 AP 311,
Departamento de Alimentos, Divisio
CP 36500 Irapuato, Guanajuato, Mexico
c
Department of Food Science, Purdue University, 745 Agricultural Mall Dr, West Lafayette, IN 47906, USA
d
Department of Science, Technology, Engineering and Mathematics Education, North Carolina State University, Raleigh, NC 27695, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2013
Received in revised form
30 April 2014
Accepted 8 May 2014
Available online 24 May 2014
The use of microwave assisted extraction (MAE) was investigated in this work for the extraction of
betalains from diced red beets. Several treatments with different combinations of time, power and duty
cycle applied to the samples were studied. The combination of 400 W and 100% duty cycle for 90e120 s
resulted in the highest amount of recovered betanines; whereas at 140e150 s the highest amount of
betaxanthins was obtained. The addition of ascorbic acid (0.040 mol/L) to the extracting solvent and the
development of a two-step MAE process with a cooling period in-between and after processing steps led
to an enhancement in the amount of pigments obtained. The effect of extraction time at each extraction
step on betalains yield was determined by applying a factorial design and surface plots were constructed.
The duration of the second step significantly affected the yield of betanines and betaxanthins obtained
(p < 0.05). A prediction model was proposed and validated to meet the optimal extraction times. Betalain
yields obtained by MAE were twice as high as those obtained during conventional extraction and conventional extraction at 80 C.
© 2014 Elsevier Ltd. All rights reserved.
Keywords:
Microwave-assisted extraction
Betalains
Red beet
Ascorbic acid
Factorial design
1. Introduction
The color of a fruit or vegetable product is one of its most
important quality attributes; however, for several processed
products the high temperatures used during thermal treatments
often result in pigment degradation and associated color losses.
One strategy currently used by some food processors includes the
addition of colorants, either synthetic or natural, to enhance,
improve or restore color (Azeredo, 2009). Synthetic dyes received
enormous media attention within the last few years due to a
possible, and still controversial, association with the development
of Attention Deficit Hyperactivity Disorder (ADHD) in susceptible
children. While the topic remains controversial, in 2010 the European Food Safety Authority required that products containing
artificial colors be labeled stating that the product “may have an
adverse effect on activity and attention in children” (Kanarek,
* Corresponding author. Tel.: þ1 (765) 496 1140.
lez).
E-mail address: fsanmartin@purdue.edu (M.F. San Martín-Gonza
http://dx.doi.org/10.1016/j.lwt.2014.05.025
0023-6438/© 2014 Elsevier Ltd. All rights reserved.
2011). This controversy along with consumer preference for natural products has recently increased the interest in natural colorants,
as they are typically perceived as healthier than artificial colorants
or as innocuous (Azeredo, 2009).
Natural colorants are ubiquitous in nature, particularly in fruits
and vegetables, however the types of natural colorants that are
commercially available remain relatively low. Examples of natural
colorants currently used include chlorophylls, carotenoids, curcuminoids, anthocyanins, betalains, bixin and carmine (Cai, Sun, &
Corke, 2003; Wissgott & Bortlik, 1996). Natural colorants have
some disadvantages over artificial ones. Typically, costs are higher
and depending on the specific colorant exhibit lower stability upon
processing and storage conditions. Therefore several research
studies have been devoted to develop ways to improve the
extractability and the stability of natural colorants.
Betalains are a type of pigments responsible for the color of
products such as red and yellow beets, prickly pears and other
cactus fruits, colored Swiss chard and amaranth grain (Frank et al.,
2005), that were studied in the 1960s and 1970s but which have
G.A. Cardoso-Ugarte et al. / LWT - Food Science and Technology 59 (2014) 276e282
not been as extensively studied as other natural colorants
(Stintzing & Carle, 2007). Betalains are stable over a pH range of
3.0e7.0 which gives them an advantage over anthocyanins, however, betalains possess low thermal stability. In red beets (Beta
vulgaris L.) the betalains comprise two main groups: betaxanthins
(e.g. vulgaxhantin I and II) and betacyanins (e.g. betanin and isobetanin), which are responsible for the yellow and violet-red hues
respectively (Gliszczynska-Swiglo, Szymusiak, & Malinowska,
2006; Kanner, Harel, & Granit, 2001); out of these, betanine, isobetanine and vulgaxanthine-I account for about 95% of the pigments in red beets (Nilsson, 1970; Wiley & Lee, 1978). As natural
colorants, betalains are approved for food coloring applications in
the United States and Europe and are exempt from certification
n, Alacid, & Ferna
ndez-Lo
pez, 2003; Dornenburg &
(Castellar, Obo
Knorr, 1996).
Extraction of natural pigments is typically done by solideliquid
extraction. Vegetable tissues are ground or macerated to facilitate
pez,
the diffusion of the pigments into the extraction solvent (Lo
rtolas, Condo
n, Raso, & Alvarez,
Pue
2009). The mechanical
disruption of the tissues is accompanied by release of cell wall
materials and other cellular components that require further purification of the extracts (Van der Poel, Schiweck, & Schwarz, 1998).
Therefore, technologies and processes capable of facilitating the
extraction from matrices as intact as possible are highly desirable
(Stintzing & Carle, 2007).
Betalains are mainly extracted through solideliquid extractions. The betalain-containing materials are macerated or ground
and pigments extracted with water, although the use of methanol
or ethanol solutions (20e50 mL/100 mL) is required to enhance
nez, & Paredes-Lo
pez, 2000).
the extraction (Delgado-Vargas, Jime
However, the shortcomings associated with conventional techniques, such as long extraction times, solvent contamination of the
product and relative low yields (Wang & Weller, 2006) have
shifted researchers interest towards the use of novel processing
techniques that improve process efficiency through enhanced
mass transfer (Tiwari & Cullen, 2012) and which are more environmentally friendly. The extraction of betalains from red beet by
several techniques such as diffusion extraction (Wiley & Lee,
1978), ultrafiltration and reverse osmosis (Lee, Wiley, Sheu, &
Schlimme, 1982), low DC electrical field, cryogenic freezing
(Zvitov, Schwartz, & Nussinovitch, 2003), aqueous two-phase
extraction (Chethana et al., 2007), pulsed electric fields
pez, Pue
rtolas,
(Chalermchat, Dejmek, & Fincan, 2004; Lo
ndez-Orte, Alvarez,
Herna
& Raso, 2009) and gamma irradiation
(Latorre, Narvaiz, Rojas, & Gerschenson, 2010; Nayak, Chethana,
Rastogi, & Raghavarao, 2006) has been reported with varying
degrees of success.
Microwave-Assisted extraction (MAE) is a promising alternative to conventional extraction (Eskilsson & Bjorklund, 2000).
Several authors have reported the advantages of MAE over conventional extraction as reduced processing time, lower solvent
and energy demand, and higher yield (Chen et al., 2008;
Hemwimon, Pavasant, & Shotipruk, 2007; Proestos & Komaitis,
2008). During microwave irradiation, the cells become thermally
stressed; as a result, the temperature and pressure inside the cell
increase to levels that result in the rupture of cell walls releasing
intracellular compounds (Pap et al., 2012). The majority of the
plant extracts obtained by MAE are dietary polyphenols, including
flavonoids, (flavons, flavonols, isoflavones, catechins, flavanones,
flavonones and cinnamic acid derivatives), anthocyanins and carotenoids (Li, Fabiano-Tixier, Abert-Vian, & Chemat, 2013). Thus,
the aim of this study was to investigate the feasibility of the
application of Microwave Assisted Extraction (MAE) in the
extraction process of betalains from red beet and to determine its
optimal extraction conditions.
277
2. Materials and methods
2.1. Raw material
Red beets (B. vulgaris L.) were purchased in a local supermarket
of West Lafayette, IN. Red beets were washed, peeled and cut in
0.5 cm thick slices that were subsequently cut into
0.5 0.5 0.5 cm3 cubes. For preliminary tests, conventional extractions were done using fresh samples; however, to reduce
variability between samples, for the rest of the experiments, 1 kg of
beet root dices were freeze-dried in a lyophilizer (The Virtis Company Inc., Gardiner NY, EEUU). For the freeze drying process, samples were frozen in liquid nitrogen and placed into the lyophilizer
for 48 h. Freeze dried samples were stored in at 18 C in screw cap
clear glass bottles flushed with nitrogen until used. The moisture
content of freeze dried diced beet root was measured by triplicate
using a Computrac MAX 2000 rapid moisture analyzer (Arizona
Instrument LLC, Chandler, AZ). Moisture content of the freeze dried
product was 2.647 ± 0.001 g/100 g dry solids.
2.2. Extraction solvent and solids to solvent ratio
A mixture of 1:1 ethanol:water and a 0.1:25 solids to solvent
ratio were selected for MAE and conventional extraction of betalain
pigments.
2.3. Microwave assisted extraction (MAE)
MAE extraction of betalains from red beets was performed using
a Microwave Accelerated Reaction System (CEM Corporation MARS,
Matthews NC, EEUU). Liquidesolid extractions were done by adding 0.1 g of the freeze dried samples to 25 mL of extraction solvent
into 125 mL teflon extraction vessels. The vessels were evenly
distributed in the vessels' rack and introduced into the cavity. Three
vessels were used each time. Selected conditions of power (400,
800 and 1200 W), duty cycle (50, 100%), time (0e160 s) with stirring were set and the extraction process started. Average temperature in the vessels was measured by infrared sensors placed inside
the microwave cavity. Immediately after the extraction process, the
red beet cubes were separated from the extraction solvent to prevent further diffusion, volume was adjusted to 25 mL if required,
and immediately cooled in an iced water bath to avoid degradation
of pigments caused by temperature. The extractions were performed one condition at a time to accurately control extraction
time and to reduce the diffusion of the pigment after the extractions during the draining of the samples. The colored extracts were
kept in closed vials and analyzed the same day of extraction. All
experiments, i.e. each extraction condition, were done in triplicate.
2.4. Two-step MAE process
After a one-step MAE was conducted, it was observed that microwaves enhanced diffusion, and the release of pigments to the
solvent was considerably higher as compared to conventional
extraction for equivalent times. However, as expected, it was also
noted that the longer the exposure of red beet dices to microwaves,
the higher the temperature increase, and thermal degradation of
extracted pigments occurred. Thus, in order to increase pigment
extraction while reducing degradation of betalains and enhancing
pigment regeneration (Han, Kim, Kim, & Kim, 1998), a two-step
MAE process was performed with addition of L-ascorbic acid
(Sigma, St Louis, MO) to the extraction solvent. A stock solution
with 1.0 mol/L was prepared. Then, an aliquot of stock solution was
used in the preparation of extraction solvent to yield L-ascorbic acid
concentrations of 0.010 and 0.040 mol/L. The two-step MAE process
278
G.A. Cardoso-Ugarte et al. / LWT - Food Science and Technology 59 (2014) 276e282
consisted in introducing a cooling step in an iced water bath for
5 min after the first extraction targeting a temperature of 23 ± 1 C
inside the vessels. This cooling step delayed degradation of betalains already extracted, and allowed for the application of a second
MAE step for further betalains extraction. The first and second MAE
steps were performed at a power level of 400 W and 100% duty
cycle. Considering the range of times at which highest extraction of
pigments was observed during the single-step MAE (90e120 s),
step duration for both extractions was between 70 and 160 s. The
combinations tested in the two-step MAE process were 70e100 s,
100e130 s, 130e100 s and 160e70 s, for the first and second
extraction respectively.
2.5. Conventional extraction
In order to compare the total amount of betalains obtained by
MAE, a conventional extraction was conducted by adding 0.1 g of
sample and 25 mL of solvent into a screw cap flask, to prevent
solvent evaporation. Flasks were subjected to continuous shaking
at a constant speed in a temperature controlled orbit-shaker (Labline Instruments, Inc., Melrose Park Ill, EEUU) at 25 C to facilitate
the diffusion of betalains into the solvent. Further conventional
extractions were performed using the same orbit-shaker at high
temperature (80 C) in order to test the effect of temperature on the
extraction yield. The temperature was selected based on the
maximum temperature reached for MAE at the longest times.
Extraction times for conventional leaching were those used for
MAE, i.e. 0e160 s with 10 s increase, and then, every 20 min for a
total extraction time of 120 min. At the end of the extraction, red
beets cubes were separated from the solvent and the colored extracts were further analyzed. Experiments were performed in
triplicate.
2.6. Maximum extractable pigment
In order to quantify the maximum amount of extractable pigments from red beets, complete extraction of betalains was conducted by mixing 0.1 g of freeze dried red beets cubes with 25 mL of
solvent and homogenizing with a high shear mixer (Polytron PT
2100, Kinematica, Luzern, Switzerland) at 20,000 rpm for 30 s.
Homogenates were transferred to 50 mL polypropylene centrifuge
tubes and centrifuged at 20,000 g (5804 Eppendorf AG, Hamburg,
Germany). Supernatants were then filtered through No. 1 Whatman
filter paper. Clear extracts were kept in closed amber vials and
betalains were quantified. Results are reported as mg of pigment/
100 g of freeze dried solids.
2.7. Quantification of betalains
Quantification of betalains was performed spectrophotometrically following the method reported by Nilsson (1970). Absorbance
of extracts was recorded using a UV/Vis spectrophotometer
(Beckman Coulter DU800, Brea CA, EEUU) at wavelengths of
537 nm, 476 nm, and 600 nm. The percentage of betanines (betanine) and betaxanthins (vulgaxhantine-I) was calculated from the
following equations:
x ¼ 1:095 ðA537 A600 Þ
(1)
y ¼ 0:258 A537 þ A476 0:742 A600
(2)
where “x” is the absorption of betanine and “y” is the absorption of
vulgaxhantine-I. Then, the concentration of betalains was calculated in mg/100 mL from BeereLambert’s Law, using E1 cm 1% ¼ 1120
pez,
for betanine and E1 cm 1% ¼ 750 for vulgaxhantine-I (Lo
rtolas, Condo
n, et al., 2009; Lo
pez, Pue
rtolas, Hern
Pue
andez-Orte,
et al., 2009; Wiley & Lee, 1978) and converted to mg/100 g of
freeze dried red beets. Absorbance of the extracts was maintained
at values <1.0, by performing volumetric dilutions when needed, to
ensure that the linear relationship between absorbance and concentration holds true.
2.8. Statistical analysis
Two-way ANOVA was done to determine the effect of extraction
method and extraction time on betalains yield, and means comparison was done by Tukey's test with a significance level at 5%.
Two-step extraction processes where power level was kept constant were analyzed by response surface analysis. A two-level
factorial design with a confidence level of 95% was analyzed using Minitab 16 software (Minitab Inc., State College, PA, EEUU).
Means of betalains yield obtained in extractions were analyzed and
the p-values of the factors and their interactions were calculated.
Surface plots were constructed to optimize the extraction conditions that maximize the yield of betanines and betaxanthins.
Finally, using the coefficients obtained in the factorial design, a
prediction model for betalains extractions yield was proposed.
3. Results and discussion
Several extraction conditions varying power level (W) and duty
cycle (%) were tested to quantify betalains yield. Due to the features
of the microwave extractor, power levels below 400 W could not be
tested, and levels above 800 W resulted in burning of the sample.
Therefore, the power and duty cycle combinations tested were
400 W/50%, 400 W/100%, 800 W/50% and 800 W/100%. Fig. 1 shows
the concentration of betanines obtained by MAE for times up to
160 s, as compared to concentrations obtained by conventional
extraction. Due to the rapid degradation of pigments and color loss,
the combination of 800 W/100% is not shown in the figure.
Highest yields of betanines were obtained with the combinations of 400 W/100% and 800 W/50%, which would deliver equivalent energy, for 100 s (128.68 and 122.90 mg of pigment/100 g of
freeze dried red beet respectively), and no significant difference
(p > 0.05) was detected between them. In all microwave treatments, extraction yields increased with increasing time until
reaching a maximum peak between 100 and 120 s, after which
yield decreased. The maximum temperature of the extracts recorded at these peaks ranged within 77.6 and 79.9 C. Enhanced
extraction yields due to microwave heating have been previously
reported for tea polyphenols and caffeine (Pan, Niu, & Liu, 2003),
artemisin (Hao, Han, Huang, Xue, & Deng, 2002), anthocyanins
(Liazid, Guerrero, Cantos, Palma, & Barroso, 2011), phenolic compounds (Ballard, Mallikarjunan, Zhou, & O’Keefe, 2010; Gallo,
Ferracane, Graziani, Ritieni, & Fogliano, 2010) and flavonoids
(Zhang et al., 2013) among others. Yield enhancement due to microwave treatment is attributed to faster heating rate during microwave exposure compared to conventional heating, and to
damage to vegetable tissue caused by microwave fields (Gao,
Huang, Moytri, & Liu, 2007; Liu, Wei, Guo, & John, 2006;
Lucchesi, Smadja, Bradshaw, Louw, & Chemat, 2007; Yansheng
et al., 2011). On the other hand, the decreasing trend observed at
longer extraction times is attributed to exposure of betalains to
high temperatures. Betalains are heat labile pigments that undergo
rapid degradation with increasing temperature and heating time
(von Elbe, Schwartz, & Hildenbrand, 1981; Herbach, Rohe,
Stintzing, & Carle, 2006; Herbach, Stintzing, & Carle, 2004, 2006).
Fig. 2 shows the amount of betaxanthins extracted under
various conditions up to 160 s as compared to conventional
extraction. The highest amounts of betaxanthins were extracted at
G.A. Cardoso-Ugarte et al. / LWT - Food Science and Technology 59 (2014) 276e282
279
Fig. 1. Concentration of betanines from red beet obtained at ( ) 400 W/100%, ( ) conventional, ( ) 800 W/50%, and ( ) 400 W/50% for up to 160 s. Error bars indicate the standard
deviation of triplicate samples.
400 W/100% for 140 and 150 s (101.41 and 100.29 mg of pigment/
100 g of freeze dried red beet respectively) and no significant difference (p > 0.05) was observed between them. Betaxanthins yield
increased with increased extraction time and maximum concentration was obtained between 140 and 150 s, with decreasing
concentrations observed at longer extraction times.
There is a noticeable difference between the times at which
maximum yields were obtained for betanines and betaxanthins.
While heat-induced chemical changes in betaxanthins are poorly
understood, recent studies on cactus pear juices demonstrated that
isomerization of indicaxhantin (a type of betaxhantine) is induced
by thermal exposure (Mobhammer, Maier, Stintzing, & Carle, 2006).
Moreover, formation of indicaxhantin by spontaneous condensation of betalamic acid released upon thermal exposure of purple
pitaya was also observed (Herbach, Rohe, et al., 2006; Herbach,
Stintzing, et al., 2006). Thus exposure to higher temperatures at
longer extraction times explains the decrease in betaxanthin
content.
When comparing MAE with conventional extractions, it is
evident that microwave treatment had a remarkable effect in the
extraction of betalains from red beets, similar to other reports
where enhanced yield and reduced extraction times for various
compounds have been reported (Ballard et al., 2010; Hao et al.,
2002; Liazid et al., 2011; Pan et al., 2003). Microwave radiation
facilitated the diffusion of pigments into the solvent during the
extraction; nonetheless, as extraction time increased, due to high
temperatures reached, degradation of pigments occurred. However, degradation of betalains occurred as a consequence of the
exposure to faster heating rate and pigment release during microwave heating as compared to conventional heating, where
heating rate was much slower. The absence of microwave effects in
betanin degradation has been recently reported (Pires-Goncalves,
Martorelli-Di Genova, Dorr, Pinto, & Leite-Bastos, 2013). Therefore, the addition of a protective compound was necessary while
betalains were exposed to microwave heating, so that, the treated
samples would be able to withstand longer irradiation times and
higher yields could be obtained.
The addition of ascorbic acid to red beets extracts has shown
beneficial effects in stabilizing betalains (Attoe & von Elbe, 1985).
However, supplementation levels are inconsistent with reported
levels between 0.003 g/100 mL and 0.2 g/100 mL (Attoe & von Elbe,
1985; Herbach, Rohe, et al., 2006; Herbach, Stintzing, et al., 2006).
However, the decrease in half-life of betanine observed at 0.1 g/
100 mL levels has been attributed to ascorbic acid acting as a prooxidant (Pasch & von Elbe, 1979). The pro-oxidant effect of ascorbic
acid seems to be dependent of product type, since in pitaya,
ascorbic acid levels of up to 1 g/100 g did not exhibit pro-oxidant
effects (Herbach, Rohe, et al., 2006; Herbach, Stintzing, et al.,
2006). In addition to concentration levels, the step at which
ascorbic acid is added seems to have an important effect. Some
studies have reported that pre-treatment addition seems to protect
the pigments better than when added after processing (Bilyk &
Howard, 1982; Han et al., 1998) and conclude that ascorbic acid
does not only aid in pigment regeneration but exerts a protective
effect during thermal processing preventing pigment degradation
(Herbach, Rohe, et al., 2006; Herbach, Stintzing, et al., 2006).
Han et al. (1998) successfully stabilized betalains through the
addition of 0.040 mol/L of various additives, including ascorbic acid,
to red beet juice subjected to thermal treatment at 100 C followed
by immediate cooling at 10 C and a regeneration time of 24 h.
Thus, in this study, in an attempt to increase extraction yields the
addition of 0.040 mol/L ascorbic acid to the solvent prior to a twostep MAE process with immediate cooling between each step and a
2 h standing time was investigated. The time for each extraction
step were chosen in the range of 70 and 160 s, based on the times
that yielded higher pigment concentrations in the absence of
ascorbic acid (~100 s). After testing several combinations of twostep extractions, four of them were selected for further comparison at lower ascorbic acid concentration, namely 0 and 0.010 mol/L.
The conditions selected were those two that showed the highest
and lowest yield and two other conditions in-between the first two.
Tables 1 and 2 show the four selected treatments and the yields of
betanines and betaxanthins obtained for the three ascorbic acid
concentrations evaluated in the two-step MAE process.
Fig. 2. Concentration of betaxanthins from red beet obtained ( ) 400 W/100%, ( ) conventional, ( ) 800 W/50%, and ( ) 400 W/50% for up to 160 s. Error bars indicate the
standard deviation of triplicate samples.
280
G.A. Cardoso-Ugarte et al. / LWT - Food Science and Technology 59 (2014) 276e282
Table 1
Yields of betanines (mg of pigment/100 g of freeze dried red beet) obtained by two-step MAE processes and varying concentration of ascorbic acid in the extraction solvent.
Concentration of ascorbic acid (mM)
Treatment 1 (70 s/100 s)
Treatment 2 (100 s/130 s)
Treatment 3 (130 s/100 s)
Treatment 4 (160 s/70 s)
40
10
0
155.86 ± 24.09aAB
156.38 ± 7.58aAB
124.04 ± 20.86aA
131.78 ± 22.89aAC
176.14 ± 9.00bA
96.22 ± 11.81cAB
187.67 ± 26.99aB
132.84 ± 30.14bAB
86.89 ± 20.68bAB
90.11 ± 5.71aC
126.28 ± 16.44bB
69.08 ± 8.94aB
Values with different lowercase letters (a, b, c) in a column indicate significant difference (p < 0.05).
Values with different uppercase letters (A, B, C) in a row indicate significant difference (p < 0.05).
Table 2
Yields of betaxanthins (mg of pigment/100 g of freeze dried red beet) obtained by two-step MAE processes and varying concentration of ascorbic acid in the extraction solvent.
Concentration of ascorbic acid (mM)
Treatment 1 (70 s/100 s)
Treatment 2 (100 s/130 s)
Treatment 3 (130 s/100 s)
Treatment 4 (160 s/70 s)
40
10
0
65.88 ± 13.51aA
81.08 ± 7.00abA
99.45 ± 16.58bA
39.37 ± 15.92aB
84.61 ± 6.18bA
117.47 ± 14.27bA
85.39 ± 2.9aA
69.66 ± 10.15aA
125.43 ± 21.75bA
59.05 ± 5.14aAB
84.23 ± 10.08aA
119.54 ± 13.70bA
Values with different lowercase letters (a, b, c) in a column indicate significant difference (p < 0.05).
Values with different uppercase letters (A, B, C) in a row indicate significant difference (p < 0.05).
The treatments which yielded the highest amount of betanines
was 130 s/100 s with 0.040 mol/L of ascorbic acid
(187.67 ± 26.99 mg/100 g red beet) and 100 s/130 s with 0.010 mol/
L ascorbic acid (176 ± 9.00 mg/100 g red beet), which showed
significant difference with respect to the other concentrations of
ascorbic acid tested (Table 1). These results suggest that ascorbic
acid added to the solvent prior to the extraction process exerts a
protective and regenerative effect on betanines during and after
repeated thermal treatments. For betaxanthins, the greatest
amount detected (125.43 ± 21.75) was obtained for the same
treatment (130 s/100 s), but without the addition of ascorbic acid
(Table 2). Regeneration of red pigments from red beets in the
absence (von Elbe et al., 1981) and presence of isoascorbic acid
(Bilyk & Howard, 1982) and other additives (Han et al., 1998) has
been reported previously. Han et al. (1998) reported that repeated
heating of a control solution with no ascorbic acid lost red pigments
continuously with no apparent regeneration, whereas in the presence of ascorbic acid, the red pigments were lost upon heating but
regenerated during storage. Pigment regeneration was reported to
be greater when the additives were added prior to heating as
opposed to adding them after the heating treatment. Furthermore,
if the pigments solutions with ascorbic acid were allowed to
regenerate after each heating step, the initial pigment concentration was maintained even after 5 heating cycles at 100 C for 3 min.
They also observed that betaxanthins were not regenerated by
ascorbic acid when comparing the absorption spectra of prepared
beet juice before and after the thermal treatment. However, a
detrimental effect of ascorbic acid on betaxanthins was not reported by Han et al. (1998). The degradation of betaxanthins has
been reported to occur faster than degradation of betanins
(Herbach et al., 2004) which would explain the lower
concentrations observed at longer heating times, however, according to our results, the presence of ascorbic acid seemed to exert
a detrimental effect on betaxanthins yield (Table 2). In order to
identify the most important factor affecting the yield of betalains
during the two-step MAE extraction and to determine the range of
extraction time to optimize the amount of pigments obtained, a
response surface analysis was carried out. Table 3 shows the pvalues and coefficients obtained for the first and second treatment
and their interactions.
The factors that significantly affected the amount of both,
betanines and betaxanthins obtained in the two-step MAE process
were the second treatment and its square. Therefore, the duration
of the 1st treatment was found to have no significant influence in
the extracted amount of betalaines, as long as it ranged within 70
and 160 s. The interaction ranges of time to optimize the yields of
betalains in the two-step MAE process are illustrated in Figs. 3 and
4 by means of response surface plots.
According to Fig. 3, to obtain the greatest amount of betanines,
the duration of the first extraction should be within the range of 70
and 100 s and the duration of the second treatment should range
between 96 and 109 s; the predicted amount of pigment extracted
should be greater than 168 mg/100 g of dried sample. Regarding
betaxanthins, as shown in Fig. 4, the combination to obtain the
highest amount of pigment is to perform the first treatment between 156 and 160 s and the second one between 92 and 120 s; the
Table 3
Coefficients and p-values for the first and second heating treatments and their
interactions.
Factors
Coefficients
p-Value
Betanines Betaxanthins Betanines Betaxanthins
Constant
74.8331 9.41864
1st treatment
0.4522 0.94207
2nd treatment
5.0781 2.50105
1st treatment1st treatment
0.0015 0.00367
2nd treatment2nd treatment 0.027 0.0139
1st treatment2nd treatment
0.0064 0.00267
a
significant factors.
e
0.329
0.043a
0.795
0.032a
0.307
e
0.181
0.045a
0.182
0.021a
0.372
Fig. 3. Response surface plot showing predicted betanines concentration (mg/100 g
freeze dried red beet) as a function of extraction time for first and second steps.
G.A. Cardoso-Ugarte et al. / LWT - Food Science and Technology 59 (2014) 276e282
Fig. 4. Response surface plot showing predicted betaxanthins concentration (mg/100 g
freeze dried red beet) as a function of extraction time for first and second steps.
amount of pigment extracted should be greater than 85 mg/100 g of
dried sample.
To validate the results obtained in the surface plots, coefficients
obtained after the response surface analysis (Table 3) were used to
construct a prediction model for betanines and betaxanthins
respectively and the accurate times of the first treatment (T) and
the second treatment (t) of the two-step MAE process to obtain the
highest yield were computed. Models were constructed as follows:
Betanines ¼ 74:83 þ ð 0:45*TÞ þ ð5:07*tÞ þ ð 0:0015*T*TÞ
þ ð 0:027*t*tÞ þ ð0:0064*T*tÞ
Betaxanthins ¼ 9:41 þ ð 0:94*TÞ þ ð2:50*tÞ þ ð0:0036*T*TÞ
þ ð 0:01*t*tÞ þ ð0:0026*T*tÞ
For betanines, the time of the first and second treatment
resulted to be 70 s and 102 s respectively, while for betaxanthins,
these times were 160 s and 106 s for the first and second steps
respectively. The conditions predicted by the model were validated
by MAE and the yields obtained were 189.02 mg/100 g of betanines
and 100.01 mg/100 g of betaxanthins.
Finally, a comparison of extraction percentage of betalains obtained with two-step MAE process, conventional extraction at room
temperature (~23 C) and conventional extraction at 80 C with
respect to the maximum extractable amounts (364.83 mg/100 g of
betanines and 195.84 mg/100 g of betaxanthins) is shown in Fig. 5.
281
Fig. 6. Concentration of betanines (black line) and betaxanthins (grey line) obtained by
conventional extraction as a function of time.
Extraction times at room or high temperature were performed for
172 and 266 s for betanines and betaxanthins respectively.
The greatest extraction percentages of betalains were obtained
with MAE for both, betanines (52%) and betaxanthins (51%), with
extraction percentages higher than 50% of the maximum extractable pigments. Conversely, the amount of pigments extracted by
conventional extraction at room or elevated temperature were
considerably lower (10e20%). Fig. 6 shows the concentration of
pigments for conventional extraction at room temperature. The
levels of pigment extracted after 60 min were equivalent to MAE for
2e3 min depending on selected conditions. Furthermore, the
extraction percentages obtained by MAE were slightly higher than
pez, Pue
rtolas, Herna
ndez-Orte, et al.,
the 46.5% reported by Lo
2009 when applying pulsed electric fields (pulses at 5 kV/cm) after 400 min of extraction at 60 C and much more higher than the
8% and 12% obtained by Thimmaraju, Bhagyalakshmi, Narayan, and
Ravishankar (2003) during a sonication-assisted pigment extraction from red beet hairy roots under continuous ultrasound of
0.02 MHz after 60 and 120 s respectively. Therefore, MAE showed
enhancement in the extraction of betalains with a remarkable
decrease in extraction time. In this work, the concentration of
betalains extracted after twenty minutes under conventional
extraction could be obtained after only two minutes using MAE.
4. Conclusions
MAE was shown to be a successful way to extract betalains from
red beet, despite of the degradation caused by the application of
heat to betalains during the process, extraction yields were higher
than conventional extraction at equivalent time and temperature.
The addition of ascorbic acid protected the extracts from degradation during processing and aided in pigment regeneration during
cooling and standing times. A two-step MAE process was proposed
in this work to further enhance extraction yields. Response surface
analysis showed that the extraction time during the second treatment influenced betalains yield the most. Thus, MAE is a promising
technology for the efficient extraction of pigments and other
bioactive compounds that warrants further investigation to
develop applications and subsequent scale up techniques.
Acknowledgments
Fig. 5. Extraction percentage from maximum extractable pigment of betanins ( ) and
betaxanthins ( ) obtained with three different methods.
Authors thank the financial support from Consejo Nacional de
xico (CONACyT) for the scholarships to
Ciencia y Tecnología, Me
author G. Cardoso-Ugarte.
282
G.A. Cardoso-Ugarte et al. / LWT - Food Science and Technology 59 (2014) 276e282
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