Accepted Manuscript

Microwave-Assisted Extraction of betalains from red beet (Beta vulgaris)

G.A. Cardoso-Ugarte , M.E. Sosa-Morales , T. Ballard , A. Liceaga , M.F. San Martín-

González

PII: S0023-6438(14)00314-4
DOI: 10.1016/j.lwt.2014.05.025
Reference: YFSTL 3925

To appear in: LWT - Food Science and Technology

Received Date: 29 August 2013

Revised Date: 30 April 2014
Accepted Date: 8 May 2014

Please cite this article as: Cardoso-Ugarte, G.A., Sosa-Morales, M.E., Ballard, T., Liceaga, A., San
Martín-González, M.F., Microwave-Assisted Extraction of betalains from red beet (Beta vulgaris), LWT -
Food Science and Technology (2014), doi: 10.1016/j.lwt.2014.05.025.

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 ACCEPTED MANUSCRIPT

1 Title:

2 Microwave-Assisted Extraction of betalains from red beet (Beta vulgaris)

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4 Authors:

5 Cardoso-Ugarte, G. A.a, Sosa-Morales, M.E.b, Ballard, T.c, Liceaga, A.c, and San

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6 Martín-González, M.F.c

a
7 Departamento de Ingeniería en Alimentos, Universidad de las Américas-Puebla,

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8 Ex-Hacienda Santa Catarina Mártir S/N, San Andrés Cholula CP 7281, Puebla,
9 México

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b
10 Departamento de Alimentos, División Ciencias de la Vida, Campus Irapuato-
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11 Salamanca, Universidad de Guanajuato, Carr. Irapuato-Silao km 9 AP 311, CP
12 36500, Irapuato, Guanajuato, México
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c
13 Department of Food Science, Purdue University, 745 Agricultural Mall Dr, West
14 Lafayette, IN, 47906, USA
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15
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16 Corresponding Author:
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17 M. Fernanda San Martin-Gonzalez

18 Department of Food Science

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19 Purdue University
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20 745 Agriculture Mall Dr

21 West Lafayette, IN 47906

22 Tel. +1 (765) 496-1140

23 fsanmartin@purdue.edu
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24 Microwave-Assisted Extraction of betalains from red beet (Beta vulgaris)

25 Cardoso-Ugarte, G. A.1, Sosa-Morales, M.E.1, Ballard, T.2, Liceaga, A.2, and San
26 Martín-González, M.F.1

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27 Departamento de Ingeniería en Alimentos, Universidad de las Américas-Puebla,

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28 Ex-Hacienda Santa Catarina Mártir S/N, San Andrés Cholula CP 7281, Puebla,
29 México

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2
30 Department of Food Science, Purdue University, 745 Agricultural Mall Dr, West
31 Lafayette, IN, 47906, USA

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32

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33 ABSTRACT
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34 The use of microwave assisted extraction (MAE) was investigated in this work for
35 the extraction of betalains from diced red beets. Several treatments with different
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36 combinations of time, power and duty cycle applied to the samples were studied.
37 The combination of 400 W and 100% duty cycle for 90-120 s resulted in the
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38 highest amount of recovered betanines; whereas at 140 - 150 s the highest amount
39 of betaxanthins was obtained. The addition of ascorbic acid (0.040 mol/L) to the
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40 extracting solvent and the development of a two-step MAE process with a cooling
41 period in-between and after processing steps led to an enhancement in the amount
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42 of pigments obtained. The effect of extraction time at each extraction step on

43 betalains yield was determined by applying a factorial design and surface plots
44 were constructed. The duration of the second step significantly affected the yield of
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45 betanines and betaxanthins obtained (p<0.05). A prediction model was proposed

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46 and validated to meet the optimal extraction times. Betalain yields obtained by
47 MAE were twice as high as those obtained during conventional extraction and
48 conventional extraction at 80°C.

49 KEYWORDS: Microwave-Assisted Extraction, betalains, red beet, ascorbic acid,

50 factorial design
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51 1. INTRODUCTION

52 The color of a fruit or vegetable product is one of its most important quality
53 attributes; however, for several processed products the high temperatures used
54 during thermal treatments often result in pigment degradation and associated color

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55 losses. One strategy currently used by some food processors includes the addition
56 of colorants, either synthetic or natural, to enhance, improve or restore color
(Azeredo, 2009). Synthetic dyes received enormous media attention within the last

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57
58 few years due to a possible, and still controversial, association with the
development of Attention Deficit Hyperactivity Disorder (ADHD) in susceptible

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59
60 children. While the topic remains controversial, in 2010 the European Food Safety
61 Authority required that products containing artificial colors be labeled stating that

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62 the product “may have an adverse effect on activity and attention in children”
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63 (Kanarek, 2011). This controversy along with consumer preference for natural
64 products has recently increased the interest in natural colorants, as they are
65 typically perceived as healthier than artificial colorants or as innocuous (Azeredo,
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66 2009).
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67 Natural colorants are ubiquitous in nature, particularly in fruits and vegetables,

68 however the types of natural colorants that are commercially available remain
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69 relatively low. Examples of natural colorants currently used include chlorophylls,

70 carotenoids, curcuminoids, anthocyanins, betalains, bixin and carmine (Cai et al.,
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71 2003; Wissgot and Botlik, 1996). Natural colorants have some disadvantages over
72 artificial ones. Typically, costs are higher and depending on the specific colorant
exhibit lower stability upon processing and storage conditions. Therefore several
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73
74 research studies have been devoted to develop ways to improve the extractability
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75 and the stability of natural colorants.

76 Betalains are a type of pigments responsible for the color of products such as red
77 and yellow beets, prickly pears and other cactus fruits, colored Swiss chard and
78 amaranth grain (Frank et al., 2005), that were studied in the 1960s and 1970s but
79 which have not been as extensively studied as other natural colorants (Stintzing &
80 Carle, 2007). Betalains are stable over a pH range of 3.0-7.0 which gives them an
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81 advantage over anthocyanins, however, betalains possess low thermal stability. In

82 red beets (Beta vulgaris L.) the betalains comprise two main groups: betaxanthins
83 (e.g. vulgaxhantin I and II) and betacyanins (e.g. betanin and isobetanin), which
84 are responsible for the yellow and violet-red hues respectively (Gliszczynska-
85 Swiglo, et al. 2006; Kanner et al., 2001); out of these, betanine, isobetanine and

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86 vulgaxanthine-I account for about 95% of the pigments in red beets (Nilsson, 1970;
87 Wiley & Lee, 1978). As natural colorants, betalains are approved for food coloring

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88 applications in the United Staes and Europe and are exempt from certification
89 (Castellar, 2003; Dornenburg et al., 1996).

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90 Extraction of natural pigments is typically done by solid-liquid extraction. Vegetable
91 tissues are ground or macerated to facilitate the diffusion of the pigments into the

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92 extraction solvent (López et al., 2009a). The mechanical disruption of the tissues is
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93 accompanied by release of cell wall materials and other cellular components that
94 require further purification of the extracts (Van der Poel et al., 1998). Therefore,
95 technologies and processes capable of facilitating the extraction from matrices as
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96 intact as possible are highly desirable (Stintzing & Carle, 2007).

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97 Betalains are mainly extracted through solid-liquid extractions. The betalain-

98 containing materials are macerated or ground and pigments extracted with water,
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99 although the use of methanol or ethanol solutions (20-50 mL/100 mL) is required to
100 enhance the extraction (Delgado-Vargas et al., 2000). However, the shortcomings
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101 associated with conventional techniques, such as long extraction times, solvent
102 contamination of the product and relative low yields (Wang & Weller, 2006) have
shifted researchers interest towards the use of novel processing techniques that
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103
104 improve process efficiency through enhanced mass transfer (Tiwari & Cullen,
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105 2012) and which are more environmentally friendly. The extraction of betalains
106 from red beet by several techniques such as diffusion extraction (Wiley & Lee,
107 1978), ultrafiltration and reverse osmosis (Lee et al., 1982), low DC electrical field,
108 cryogenic freezing (Zvitov et al., 2003), aqueous two-phase extraction (Chethana
109 et al., 2007), pulsed electric fields (Chalermchat et al.,2004; López et al., 2009b)
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110 and gamma irradiation (Nayak et al., 2006; Latorre et al., 2010) has been reported
111 with varying degrees of success.

112 Microwave-Assisted extraction (MAE) is a promising alternative to conventional

113 extraction (Eskilsson & Bjorklund, 2000). Several authors have reported the

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114 advantages of MAE over conventional extraction as reduced processing time,
115 lower solvent and energy demand, and higher yield (Hemwimon et al., 2007;
Proestos & Komaitis, 2008; Chen et al., 2008). During microwave irradiation, the

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116
117 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

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118
119 intracellular compounds (Pap et al., 2012). The majority of the plant extracts
120 obtained by MAE are dietary polyphenols, including flavonoids, (flavons, flavonols,

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121 isoflavones, catechins, flavanones, flavonones and cinnamic acid derivatives),
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122 anthocyanins and carotenoids (Li et al., 2013). Thus, the aim of this study was to
123 investigate the feasibility of the application of Microwave Assisted Extraction (MAE)
124 in the extraction process of betalains from red beet and to determine its optimal
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125 extraction conditions.

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126 2. MATERIALS AND METHODS

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127 2.1 Raw material

128 Red beets (Beta vulgaris L.) were purchased in a local supermarket of West
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129 Lafayette, IN. Red beets were washed, peeled and cut in 0.5 cm thick slices that
130 were subsequently cut into 0.5 x 0.5 x 0.5 cm3 cubes. For preliminary tests,
131 conventional extractions were done using fresh samples; however, to reduce
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132 variability between samples, for the rest of the experiments, 1 kg of beet root dices
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133 were freeze-dried in a lyophilizer (The Virtis Company Inc., Gardiner NY, EEUU).
134 For the freeze drying process, samples were frozen in liquid nitrogen and placed
135 into the lyophilizer for 48 h. Freeze dried samples were stored in at -18oCin screw
136 cap clear glass bottles flushed with nitrogen until used. The moisture content of
137 freeze dried diced beet root was measured by triplicate using a Computrac MAX
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138 2000 rapid moisture analyzer (Arizona Instrument LLC, Chandler, AZ). Moisture
139 content of the freeze dried product was 2.647 ± 0.001 g / 100 g dry solids.

140 2.2 Extraction solvent and solids to solvent ratio

141 A mixture of 1:1 ethanol:water and a 0.1:25 solids to solvent ratio were selected for

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142 MAE and conventional extraction of betalain pigments.

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143

144 2.3 Microwave assisted extraction (MAE)

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145 MAE extraction of betalains from red beets was performed using a Microwave
146 Accelerated Reaction System (CEM Corporation MARS, Matthews NC, EEUU).

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147 Liquid-solid extractions were done by adding 0.1 g of the freeze dried samples to
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148 25 mL of extraction solvent into 125 mL teflon extraction vessels. The vessels were
149 evenly distributed in the vessels’ rack and introduced into the cavity. Three vessels
150 were used each time. Selected conditions of power (400, 800 and 1200 W), duty
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151 cycle (50, 100%), time (0-160s) with stirring were set and the extraction process
152 started. Average temperature in the vessels was measured by infrared sensors
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153 placed inside the microwave cavity. Immediately after the extraction process, the
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154 red beet cubes were separated from the extraction solvent to prevent further
155 diffusion, volume was adjusted to 25mL if required, and immediately cooled in an
156 iced water bath to avoid degradation of pigments caused by temperature. The
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157 extractions were performed one condition at a time to accurately control extraction
158 time and to reduce the diffusion of the pigment after the extractions during the
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159 draining of the samples. The coloured extracts were kept in closed vials and
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160 analyzed the same day of extraction. All experiments, i.e. each extraction
161 condition, were done in triplicate.

162 2.4 Two-step MAE process

163 After a one-step MAE was conducted, it was observed that microwaves enhanced
164 diffusion, and the release of pigments to the solvent was considerably higher as
165 compared to conventional extraction for equivalent times. However, as expected, it
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166 was also noted that the longer the exposure of red beet dices to microwaves, the
167 higher the temperature increase, and thermal degradation of extracted pigments
168 occurred. Thus, in order to increase pigment extraction while reducing degradation
169 of betalains and enhancing pigment regeneration (Han et al., 1998), a two-step
170 MAE process was performed with addition of L-ascorbic acid (Sigma, St Louis,

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171 MO) to the extraction solvent. A stock solution with 1.0 mol/L was prepared. Then,
172 an aliquot of stock solution was used in the preparation of extraction solvent to

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173 yield L-ascorbic acid concentrations of 0.010 and 0.040 mol/L. The two-step MAE
174 process consisted in introducing a cooling step in an iced water bath for 5 min after

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175 the first extraction targeting a temperature of 23±1°C inside the vessels. This
176 cooling step delayed degradation of betalains already extracted, and allowed for

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177 the application of a second MAE step for further betalains extraction. The first and
178 second MAE steps were performed at a power level of 400 W and 100% duty
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179 cycle. Considering the range of times at which highest extraction of pigments was
180 observed during the single-step MAE (90 to 120s), step duration for both
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181 extractions was between 70 and 160s. The combinations tested in the two-step
182 MAE process were 70-100 s, 100-130 s, 130-100 s and 160-70 s, for the first and
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183 second extraction respectively.

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184 2.5 Conventional extraction

185 In order to compare the total amount of betalains obtained by MAE, a conventional
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186 extraction was conducted by adding 0.1 g of sample and 25 mL of solvent into a
187 screw cap flask, to prevent solvent evaporation. Flasks were subjected to
continuous shaking at a constant speed in a temperature controlled orbit-shaker
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188
189 (Lab-line Instruments, Inc., Melrose Park Ill, EEUU) at 25oC to facilitate the
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190 diffusion of betalains into the solvent. Further conventional extractions were
191 performed using the same orbit-shaker at high temperature (80oC) in order to test
192 the effect of temperature on the extraction yield. The temperature was selected
193 based on the maximum temperature reached for MAE at the longest times.
194 Extraction times for conventional leaching were those used for MAE, i.e. 0 to 160 s
195 with 10 s increase, and then, every 20 min for a total extraction time of 120 min. At
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196 the end of the extraction, red beets cubes were separated from the solvent and the
197 colored extracts were further analyzed. Experiments were performed in triplicate.

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199 2.6 Maximum extractable pigment

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200 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

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202 beets cubes with 25 mL of solvent and homogenizing with a high shear mixer
203 (Polytron PT 2100, Kinematica, Luzern, Switzerland) at 20,000 RPM for 30s.

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204 Homogenates were transferred to 50 mL polypropylene centrifuge tubes and
205 centrifuged at 20,000 x g (5804 Eppendorf AG, Hamburg, Germany).

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206 Supernatants were then filtered through No. 1 Whatman filter paper. Clear extracts
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207 were kept in closed amber vials and betalains were quantified. Results are
208 reported as mg of pigment / 100 g of freeze dried solids).
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209 2.7 Quantification of betalains

210 Quantification of betalains was performed spectrophotometrically following the

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211 method reported by Nilsson (1970). Absorbance of extracts was recorded using a
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212 UV/Vis spectrophotometer (Beckman Coulter DU800, Brea CA, EEUU) at

213 wavelengths of 537 nm, 476 nm, and 600 nm. The percentage of betanines
214 (betanine) and betaxanthins (vulgaxhantine-I) was calculated from the following
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215 equations:

216 x = 1.095 (A537 – A600) [1]

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217 y = -0.258 A537 + A476 – 0.742 A600 [2]

218 where “x” is the absorption of betanine and “y” is the absorption of vulgaxhantine-I.
219 Then, the concentration of betalains was calculated in mg/100 mL from Beer-
220 Lambert’s Law, using E1cm1% = 1120 for betanine and E1cm1% = 750 for
221 vulgaxhantine-I (Wiley & Lee, 1978; López et al., 2009a; López et al., 2009b) and
222 converted to mg/100 g of freeze dried red beets. Absorbance of the extracts was
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223 maintained at values < 1.0, by performing volumetric dilutions when needed, to
224 ensure that the linear relationship between absorbance and concentration holds
225 true.

226

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227 2.8 Statistical analysis

Two-way ANOVA was done to determine the effect of extraction method and

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228
229 extraction time on betalains yield, and means comparison was done by Tukey’s
230 test with a significance level at 5%. Two-step extraction processes where power

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231 level was kept constant were analyzed by response surface analysis. A two-level
232 factorial design with a confidence level of 95% was analyzed using Minitab 16

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233 software (Minitab Inc., State College, PA, EEUU). Means of betalains yield
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234 obtained in extractions were analyzed and the p-values of the factors and their
235 interactions were calculated. Surface plots were constructed to optimize the
236 extraction conditions that maximize the yield of betanines and betaxanthins.
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237 Finally, using the coefficients obtained in the factorial design, a prediction model for
238 betalains extractions yield was proposed.
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239
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240 3. RESULTS AND DISCUSSION

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241 Several extraction conditions varying power level (W) and duty cycle (%) were
242 tested to quantify betalains yield. Due to the features of the microwave extractor,
243 power levels below 400 W could not be tested, and levels above 800 W resulted in
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244 burning of the sample. Therefore, the power and duty cycle combinations tested
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245 were 400W/50%, 400W/100%, 800W/50% and 800W/100%. Figure 1 shows the
246 concentration of betanines obtained by MAE for times up to 160 seconds, as
247 compared to concentrations obtained by conventional extraction. Due to the rapid
248 degradation of pigments and color loss, the combination of 800W/100% is not
249 shown in the figure.
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250 Highest yields of betanines were obtained with the combinations of 400W/100%
251 and 800W/50%, which would deliver equivalent energy, for 100 s (128.68 and
252 122.90 mg of pigment / 100 g of freeze dried red beet respectively), and no
253 significant difference (p>0.05) was detected between them. In all microwave
254 treatments, extraction yields increased with increasing time until reaching a

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255 maximum peak between 100 and 120 s, after which yield decreased. The
256 maximum temperature of the extracts recorded at these peaks ranged within 77.6

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257 and 79.9°C. Enhanced extraction yields due to micro wave heating have been
258 previously reported for tea polyphenols and caffeine (Pan et al., 2003), artemisin

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259 (Hao et al., 2002), anthocyanins (Liazid et al., 2011), phenolic compounds (Ballard
260 et al., 2010; Gallo et al., 2010) and flavonoids (Zhang et al., 2013) among others.

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261 Yield enhancement due to microwave treatment is attributed to faster heating rate
262 during microwave exposure compared to conventional heating, and to damage to
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263 vegetable tissue caused by microwave fields (Gao et al., 2007; Liu et al., 2006;
264 Lucchesi et al., 2007; Yansheng et al., 2011). On the other hand, the decreasing
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265 trend observed at longer extraction times is attributed to exposure of betalains to

266 high temperatures. Betalains are heat labile pigments that undergo rapid
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267 degradation with increasing temperature and heating time (Von Elbe et al., 1981;
268 Herbach et al., 2004; Herbach et al., 2006).
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269 Figure 2 shows the amount of betaxanthins extracted under various conditions up
270 to 160 s as compared to conventional extraction. The highest amounts of
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271 betaxanthins were extracted at 400W/100% for 140 and 150 s (101.41 and 100.29
272 mg of pigment / 100 g of freeze dried red beet respectively) and no significant
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273 difference (p>0.05) was observed between them. Betaxanthins yield increased with
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274 increased extraction time and maximum concentration was obtained between 140
275 and 150 s, with decreasing concentrations observed at longer extraction times.

276 There is a noticeable difference between the times at which maximum yields were
277 obtained for betanines and betaxanthins. While heat-induced chemical changes in
278 betaxanthins are poorly understood, recent studies on cactus pear juices
279 demonstrated that isomerization of indicaxhantin (a type of betaxhantine) is
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280 induced by thermal exposure (Moβhammer et al., 2006). Moreover, formation of

281 indicaxhantin by spontaneous condensation of betalamic acid released upon
282 thermal exposure of purple pitaya was also observed (Herbach et al., 2006). Thus
283 exposure to higher temperatures at longer extraction times explains the decrease
284 in betaxanthin content.

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285 When comparing MAE with conventional extractions, it is evident that microwave

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286 treatment had a remarkable effect in the extraction of betalains from red beets,
287 similar to other reports where enhanced yield and reduced extraction times for

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288 various compounds have been reported (Hao et al., 2002; Pan et al., 2003; Ballard
289 et al., 2010; Liazid et al., 2011). Microwave radiation facilitated the diffusion of
290 pigments into the solvent during the extraction; nonetheless, as extraction time

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291 increased, due to high temperatures reached, degradation of pigments occurred.
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292 However, degradation of betalains occurred as a consequence of the exposure to
293 faster heating rate and pigment release during microwave heating as compared to
294 conventional heating, where heating rate was much slower. The absence of
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295 microwave effects in betanin degradation has been recently reported (Pires-
296 Goncalves et al., 2013). Therefore, the addition of a protective compound was
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297 necessary while betalains were exposed to microwave heating, so that, the treated
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298 samples would be able to withstand longer irradiation times and higher yields could
299 be obtained.
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300 The addition of ascorbic acid to red beets extracts has shown beneficial effects in
301 stabilizing betalains (Attoe & von Elbe, 1985). However, supplementation levels are
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302 inconsistent with reported levels between 0.003 g/100mL and 0.2 g/100mL (Attoe
303 & von Elbe, 1985; Herbach et al., 2006). However, the decrease in half-life of
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304 betanine observed at 0.1g/100mL levels has been attributed to ascorbic acid acting
305 as a pro-oxidant (Pasch & von Elbe, 1979). The pro-oxidant effect of ascorbic acid
306 seems to be dependent of product type, since in pitaya, ascorbic acid levels of up
307 to 1 g/100g did not exhibit pro-oxidant effects (Herbach et al., 2006). In addition to
308 concentration levels, the step at which ascorbic acid is added seems to have an
309 important effect. Some studies have reported that pre-treatment addition seems to
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310 protect the pigments better than when added after processing (Bilyk & Howard,
311 1982; Han et al., 1998) and conclude that ascorbic acid does not only aid in
312 pigment regeneration but exerts a protective effect during thermal processing
313 preventing pigment degradation (Herbach et al., 2006).

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314 Han et al. (1998) successfully stabilized betalains through the addition of 0.040
315 mol/L of various additives, including ascorbic acid, to red beet juice subjected to
thermal treatment at 100°C followed by immediate co oling at 10°C and a

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317 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 two-step

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319 MAE process with immediate cooling between each step and a 2 h standing time
320 was investigated. The time for each extraction step were chosen in the range of 70

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321 and 160 s, based on the times that yielded higher pigment concentrations in the
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322 absence of ascorbic acid (~100s). After testing several combinations of two-step
323 extractions, four of them were selected for further comparison at lower ascorbic
324 acid concentration, namely 0 and 0.010 mol/L. The conditions selected were those
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325 two that showed the highest and lowest yield and two other conditions in-between
326 the first two. Tables 1 and 2 show the four selected treatments and the yields of
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327 betanines and betaxanthins obtained for the three ascorbic acid concentrations
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328 evaluated in the two-step MAE process.

329
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330 The treatments which yielded the highest amount of betanines was 130s/100s with
331 0.040 mol/L of ascorbic acid (187.67±26.99 mg/100g red beet) and 100s/130s with
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332 0.010 mol/L ascorbic acid (176±9.00 mg/100g red beet) , which showed significant
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333 difference with respect to the other concentrations of ascorbic acid tested (Table
334 1). These results suggest that ascorbic acid added to the solvent prior to the
335 extraction process exerts a protective and regenerative effect on betanines during
336 and after repeated thermal treatments. For betaxanthins, the greatest amount
337 detected (125.43±21.75) was obtained for the same treatment (130s/100s), but
338 without the addition of ascorbic acid (Table 2). Regeneration of red pigments from
339 red beets in the absence (Von Elbe et al., 1981) and presence of isoascorbic acid
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340 (Bilyk & Howard, 1982) and other additives (Han et al., 1998) has been reported
341 previously. Han et al. (1998) reported that repeated heating of a control solution
342 with no ascorbic acid lost red pigments continuously with no apparent
343 regeneration, whereas in the presence of ascorbic acid, the red pigments were lost
344 upon heating but regenerated during storage. Pigment regeneration was reported

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345 to be greater when the additives were added prior to heating as opposed to adding
346 them after the heating treatment. Furthermore, if the pigments solutions with

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347 ascorbic acid were allowed to regenerate after each heating step, the initial
348 pigment concentration was maintained even after 5 heating cycles at 100°C for 3

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349 min. They also observed that betaxanthins were not regenerated by ascorbic acid
350 when comparing the absorption spectra of prepared beet juice before and after the

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351 thermal treatment. However, a detrimental effect of ascorbic acid on betaxanthins
352 was not reported by Han et al. (1998). The degradation of betaxanthins has been
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353 reported to occur faster than degradation of betanins (Herbach et al., 2004) which
354 would explain the lower concentrations observed at longer heating times, however,
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355 according to our results, the presence of ascorbic acid seemed to exert a
356 detrimental effect on betaxanthins yield (Table 2). In order to identify the most
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357 important factor affecting the yield of betalains during the two-step MAE extraction
358 and to determine the range of extraction time to optimize the amount of pigments
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359 obtained, a response surface analysis was carried out. Table 3 shows the p-values
360 and coefficients obtained for the first and second treatment and their interactions.
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361 The factors that significantly affected the amount of both, betanines and
362 betaxanthins obtained in the two-step MAE process were the second treatment
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363 and its square. Therefore, the duration of the 1st treatment was found to have no
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364 significant influence in the extracted amount of betalaines, as long as it ranged

365 within 70 and 160 s. The interaction ranges of time to optimize the yields of
366 betalains in the two-step MAE process are illustrated in Figures 3 and 4 by means
367 of response surface plots.

368 According to Figure 3, to obtain the greatest amount of betanines, the duration of
369 the first extraction should be within the range of 70 and 100 s and the duration of
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370 the second treatment should range between 96 to 109 s; the predicted amount of
371 pigment extracted should be greater than 168 mg/100 g of dried sample.
372 Regarding betaxanthins, as shown in Figure 4, the combination to obtain the
373 highest amount of pigment is to perform the first treatment between 156 and 160 s
374 and the second one between 92 to 120 s; the amount of pigment extracted should

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375 be greater than 85 mg/100 g of dried sample.

To validate the results obtained in the surface plots, coefficients obtained after the

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376
377 response surface analysis (Table 3) were used to construct a prediction model for
betanines and betaxanthins respectively and the accurate times of the first

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378
379 treatment (T) and the second treatment (t) of the two-step MAE process to obtain
380 the highest yield were computed. Models were constructed as follows:

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382 Betanines= -74.83 + (-0.45*T) + (5.07*t) + (-0.0015*T*T) + (-0.027*t*t) + (0.0064*T*t)
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383 Betaxanthins= -9.41 + (-0.94*T) + (2.50*t) + (0.0036*T*T) + (-0.01*t*t) + (0.0026*T*t)

384
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385 For betanines, the time of the first and second treatment resulted to be 70 s and
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386 102 s respectively, while for betaxanthins, these times were 160 s and 106 s for
387 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
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388
389 and 100.01 mg/100 g of betaxanthins.
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390 Finally, a comparison of extraction percentage of betalains obtained with two-step

391 MAE process, conventional extraction at room temperature (~23oC) and
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392 conventional extraction at 80°C with respect to the maximum extractable amounts
393 (364.83 mg/100 g of betanines and 195.84 mg/100 g of betaxanthins) is shown in
394 Figure 5. Extraction times at room or high temperature were performed for 172 and
395 266s for betanines and betaxanthins respectively.

396 The greatest extraction percentages of betalains were obtained with MAE for both,
397 betanines (52%) and betaxanthins (51%), with extraction percentages higher than
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398 50% of the maximum extractable pigments. Conversely, the amount of pigments
399 extracted by conventional extraction at room or elevated temperature were
400 considerably lower (10-20%). Fig. 6 shows the concentration of pigments for
401 conventional extraction at room temperature. The levels of pigment extracted after
402 60 min were equivalent to MAE for 2-3 min depending on selected conditions.

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403 Furthermore, the extraction percentages obtained by MAE were slightly higher than
404 the 46.5% reported by López et al. (2009b) when applying pulsed electric fields

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405 (pulses at 5 kV/cm) after 400 min of extraction at 60°C and much more higher than
406 the 8% and 12% obtained by Thimmaraju et al. (2003) during a sonication-assisted

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407 pigment extraction from red beet hairy roots under continuous ultrasound of 0.02
408 MHz after 60 and 120 s respectively. Therefore, MAE showed enhancement in the

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409 extraction of betalains with a remarkable decrease in extraction time. In this work,
410 the concentration of betalains extracted after twenty minutes under conventional
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411 extraction could be obtained after only two minutes using MAE.

412 4. CONCLUSIONS
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413 MAE was shown to be a successful way to extract betalains from red beet, despite
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414 of the degradation caused by the application of heat to betalains during the
415 process, extraction yields were higher than conventional extraction at equivalent
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416 time and temperature. The addition of ascorbic acid protected the extracts from
417 degradation during processing and aided in pigment regeneration during cooling
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418 and standing times. A two-step MAE process was proposed in this work to further
419 enhance extraction yields. Response surface analysis showed that the extraction
time during the second treatment influenced betalains yield the most. Thus, MAE is
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420
421 a promising technology for the efficient extraction of pigments and other bioactive
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422 compounds that warrants further investigation to develop applications and

423 subsequent scale up techniques.

424 ACKNOWLEDGMENTS

425 Authors thank the financial support from Consejo Nacional de Ciencia y
426 TecnologÍa, México (CONACyT) for the scholarships to author G. Cardoso-Ugarte.
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427
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Figure 1. Concentration of betanines from red beet obtained at ( ) 400W/100%, ( )

conventional, ( ) 800W/50%, and ( ) 400W/50% for up to 160 s. Error bars

indicate the standard deviation of triplicate samples.

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Figure 2. Concentration of betaxanthins from red beet obtained ( ) 400W/100%, ( )

conventional, ( ) 800W/50%, and ( ) 400W/50% for up to 160 s. Error bars

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indicate the standard deviation of triplicate samples.

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Figure 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.

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Figure 4. Response surface plot showing predicted betaxanthins concentration (mg /
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100 g freeze dried red beet) as a function of extraction time for first and second steps.
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Figure 5. Extraction percentage from maximum extractable pigment of betanins ( )

and betaxanthins ( ) obtained with three different methods.

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Figure 6. Concentration of betanines (black line) and betaxanthins (grey line) obtained

by conventional extraction as a function of time.

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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.

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Concentration
Treatment 1 Treatment 2 Treatment 3 Treatment 4
of ascorbic
(70s/100s) (100s/130s) (130s/100s) (160s/70s)

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acid (mM)
aAC aB aC
40 155.86±24.09aAB 131.78±22.89 187.67±26.99 90.11±5.71

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aAB bA bB
10 156.38±7.58 176.14±9.00 132.84±30.14bAB 126.28±16.44
aA cAB aB
0 124.04±20.86 96.22±11.81 86.89±20.68bAB 69.08±8.94

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Values with different lowercase letters (a, b, c) in a column indicate significant difference (p<0.05)
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Values with different uppercase letters (A, B, C) in a row indicate significant difference (p<0.05)
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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.

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Concentration
Treatment 1 Treatment 2 Treatment 3 Treatment 4
of ascorbic
(70s/100s) (100s/130s) (130s/100s) (160s/70s)

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acid (mM)
aA aB aA aAB
40 65.88±13.51 39.37±15.92 85.39±2.9 59.05±5.14

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abA bA aA aA
10 81.08±7.00 84.61±6.18 69.66±10.15 84.23±10.08
bA bA bA bA
0 99.45±16.58 117.47±14.27 125.43±21.75 119.54±13.70

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Table 3. Coefficients and p-values for the first and second heating treatments and their

interactions

Factors Coefficients p-value

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Betanines Betaxanthins Betanines Betaxanthins

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Constant -74.8331 -9.41864 ------- -------

1st treatment
-0.4522 -0.94207 0.329 0.181

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2nd treatment
5.0781 2.50105 0.043* 0.045*

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1st treatment*1st
-0.0015 0.00367 0.795 0.182
treatment
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2nd treatment*2nd
-0.027 -0.0139 0.032* 0.021*
treatment
1st treatment*2nd
0.0064 0.00267 0.307 0.372
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*
significant factors
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FIG. 6

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Amount of pigment extracted (mg/100g)

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HIGHLIGHTS:

1. Microwave-Assisted Extraction (MAE) is an efficient technology for betalain extraction.

2. Yields obtained after 2 min of MAE were comparable to conventional leaching for 20 min

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3. At equivalent times, MAE yield was 5-fold greater than conventional leaching yield.

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