Manuscript Details

Manuscript number FBIO_2017_594

Title Ultrasound-assisted extraction of carotenoids from mango (Mangifera indica L.

‘Ataulfo’) by-products on in vitro bioaccessibility

Abstract
Mango by-products (peel and paste) are a source of carotenoids, but their bioaccessibility (BA) can be limited by
dietary fiber (DF), since it retains these compounds within its structure. The aim of this study was to evaluate the
influence of ultrasound-assisted extraction (UAE) on the BA of carotenoids by an in vitro digestion model. The β-
cryptoxanthin (βCr) content (3.59 ± 0.43 mg/g DW) in UAE-peel was higher than that in Control-peel (0.66 ± 0.03 mg/g
DW). The β-carotene (βC) content in UAE-peel and UAE-paste was higher than that in Control-peel and Control-paste.
The %BA in the UAE-peel improved by 46.93%, 35.21% and 32.62% for βCr, Lutein (Lut), and βC, respectively,
compared to that in the Control-peel, and in the UAE-paste, the treatment improved the %BA for Lut, βCr, and βC by
46.04%, 44.16%, and 44.01% respectively, compared with that of the Control-paste. A high percentage of non-
bioaccessible βC was shown for the Control-peel (79.48%) and Control-paste (70.41%), and the percentage was lower
in the UAE samples. The released carotenoids were quantified in a kinetic model, and β-Cr, Lut, and βC were
effectively released in mango UAE-peel. The constant release rate, k, did not show significant differences in both
samples. A 2-parameter non-linear regression model was the best fit for the release kinetics. The use of UAE
noticeably improved the bioaccessibility of carotenoids in mango by-products.

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Highlights

 Ultrasound assisted extraction (UAE) improved carotenoids yield in Mango by-

product

 βCr from UAE-peel was the most bioaccessible with 46.93%

 UAE-paste increased the bioaccessibility of carotenoids during digestion

 β-cryptoxanthin, lutein, and β-carotene were effectively released in mango UAE-

peel
1 Ultrasound-assisted extraction of carotenoids from mango (Mangifera indica L.

2 ‘Ataulfo’) by-products on in vitro bioaccessibility

3 Short Title: Ultrasound-assisted extraction of carotenoids from mango

4 Gilberto Mercado-Mercado1, Efigenia Montalvo-González1, Gustavo A González-Aguilar2

5 Emilio Alvarez-Parrilla3and Sonia G Sáyago-Ayerdi1*

7 1 Laboratorio Integral de Investigación en Alimentos, División de Estudios de Posgrado,

8 Instituto Tecnológico de Tepic, Av. Tecnológico 2595, Postal Code 63175, Tepic, Nayarit,

9 México.

10 2Laboratorio de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y

11 Desarrollo A.C., Carretera a La Victoria Km 0.6 Postal Code 83304, Hermosillo, Sonora,

12 México.

13 3Departamento de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas,

14 Universidad Autónoma de Ciudad Juárez, Anillo Envolvente del PRONAF y Estocolmo,

15 s/n

16 Ciudad Juárez, Chih Postal Code 32310, México.

17

18

19

20 *Corresponding author: SG Sáyago-Ayerdi.

21 Tel: (+52) 311 112 0824

22 E-mail: sonia.sayago@gmail.com; ssayago@ittepic.edu.mx

23

1
24 ABSTRACT

25 Mango by-products (peel and paste) are a source of carotenoids, but their bioaccessibility

26 (BA) can be limited by dietary fiber (DF), since it retains these compounds within its

27 structure. The aim of this study was to evaluate the influence of ultrasound-assisted

28 extraction (UAE) on the BA of carotenoids by an in vitro digestion model. The β-

29 cryptoxanthin (βCr) content (3.59 ± 0.43 mg/g DW) in UAE-peel was higher than that in

30 Control-peel (0.66 ± 0.03 mg/g DW). The β-carotene (βC) content in UAE-peel and UAE-

31 paste was higher than that in Control-peel and Control-paste. The %BA in the UAE-peel

32 improved by 46.93%, 35.21% and 32.62% for βCr, Lutein (Lut), and βC, respectively,

33 compared to that in the Control-peel, and in the UAE-paste, the treatment improved the

34 %BA for Lut, βCr, and βC by 46.04%, 44.16%, and 44.01% respectively, compared with

35 that of the Control-paste. A high percentage of non-bioaccessible βC was shown for the

36 Control-peel (79.48%) and Control-paste (70.41%), and the percentage was lower in the

37 UAE samples. The released carotenoids were quantified in a kinetic model, and β-Cr, Lut,

38 and βC were effectively released in mango UAE-peel. The constant release rate, k, did not

39 show significant differences in both samples. A 2-parameter non-linear regression model

40 was the best fit for the release kinetics. The use of UAE noticeably improved the

41 bioaccessibility of carotenoids in mango by-products.

42

43 Keywords: ultrasound assisted extraction; carotenoids; bioaccessibility; mango by-

44 products; kinetics release

45

46

47

2
48 1. Introduction

49 Currently, there is growing interest in the extraction of bioactive compounds in fruit by-

50 products (BPs) (Rafiq et al., 2016). This idea is supported because consumers are

51 concerned about the safety of synthetic additives and their negative health effects (Ajila et

52 al., 2013). Recent studies have shown that BP of mango (Mangifera indica L. ‘Ataulfo’) are

53 a good source of dietary fiber (DF) and bioactive compounds, such as polyphenols and

54 carotenoids (Ajila et al. 2013; Blancas-Benítez et al. 2015). Carotenoids are responsible for

55 the colors in certain fruits and vegetables. To date, thirty-three different carotenoids have

56 been identified in mango peel, including antheraxanthin, α-carotene, violaxanthin, cis-

57 isomers, lutein epoxy derivatives, β-carotene, and others (Ornelas-Pas et al. 2007; Kiokias

58 et al. 2016). In the industrial processing of mango, the pulp is squeezed to obtain the

59 concentrate and a BP called paste is obtained. In this BP, polyphenols and DF have been

60 quantified (Cano and De Ancos 1997; Blancas-Benítez et al., 2015). However, adequate

61 techniques or extraction methods that avoid degradation or affect its biological activity are

62 required to obtain the maximum amounts of these compounds (Gil-Chávez et al., 2013).

63 Nevertheless, the utilization of carotenoids in food is very limited because of their low

64 bioaccessibility (BA). BA refers to the fraction of a compound that becomes available for

65 absorption in the small intestine (Estévez-Santiago et al., 2016; Chacón-Ordóñez et al.,

66 2016). Previous studies demonstrated that the BA of carotenoids is determined by their

67 chemical structure, molecular size, hydrophobicity, solubility, pKa, interactions with the

68 food matrix, and conjugation with other components of the diet (van Buggenhout et al.,

69 2010; Zhai et al., 2016). For this reason, emerging technologies, such as ultrasound-assisted

70 extraction (UAE), have been used to increase the BA of these and other compounds. UAE

71 can release high quantities of carotenoids due to the rupture of cell walls by cavitation

3
72 (Anesse et al., 2015; Gille et al., 2016). Information regarding the effect of UAE on the

73 bioaccessibility of carotenoids in the BP of tropical fruit is scarce. Therefore, the aim of

74 this study was to apply UAE to mango ‘Ataulfo’ BPs (peel and paste) in order to release the

75 carotenoids and evaluate their BA, which was analyzed using an in vitro digestion model.

76

77 2. Materials and Methods

78 2.1 Reagents

79 Standards of β-carotene (Type I, approx. 95% UV), β-cryptoxanthin, lutein powder (purity

80 >95%); butylated hydroxytoluene (BHT), sodium chloride, tris-maleate, porcine pepsin

81 (300 IU/mg), porcine pancreatin (P1750) and α-amylase from porcine pancreas (A-3176)

82 were purchased from Sigma-Aldrich, St. Louis, Missouri, USA. Tetrahydrofuran was

83 purchased from Wako-Pure-Chemical-Industries, Limited Company. Methanol,

84 acetonitrile, ethyl acetate, sodium phosphate dibasic anhydrous, and sodium phosphate

85 monobasic monohydrate (HPLC grade) were purchased from Fisher-Scientific, Fairlawn,

86 New Jersey, USA.

87 2.2 Sample preparation

88 Mango ‘Ataulfo’ BP (peels and paste) were obtained from a local industry (Mexifrutas,

89 S.A. de C.V. Tepic Nayarit, Mexico) during the processing of mango to obtain mango

90 concentrate and were transported to the Technological Institute of Tepic. Peels and paste

91 were washed with tap water (25°C), dried in an oven (60°C, Scorpion Scientific, A-52055,

92 Mexico), ground (IKA®, Werke, M20S3, USA), sieved in a mesh (1 mm), and stored at -

93 20°C until analysis. One batch was used as Control samples, and the other one was

94 submitted to ultrasound-assisted extraction (UAE) treatment.

95 2.3 Ultrasound-assisted extraction (UAE) treatment in mango by-products

4
96 The ultrasound device consists of a transducer for converting electric signals into ultrasonic

97 waves and a probe with a sonotrode, a tip (diameter of 7 mm), and an acoustic power

98 density of 300 W/cm2. The maximal nominal output power of the device (UP 400S,

99 Hielscher Company, Teltow, Germany) was 400 W, and the ultrasonic frequency was 24

100 kHz. The optimal UAE conditions applied to the samples were determined in a previous

101 study in our laboratory: extraction time (30 min), duty cycle (0.8), and sonication amplitude

102 (30%). Samples (300 mg) were mixed with BHT (0.1 mg/g of sample) and HCl–KCl buffer

103 (10 ml, 0.2 M, pH 1.5), and placed in a cold water bath (5°C ± 2), and the ultrasonic

104 transducer was immersed 20 mm from the bottom of a beaker using an acoustic energy

105 density of 9 W/ml. This value corresponds to 90 W of ultrasound power or 30% of the

106 amplitude used. After the UAE treatment, an in vitro digestion was performed.

107 2.4 In vitro digestion model and bioaccessibility of carotenoids in mango by-products

108 An in vitro digestion procedure according to Saura-Calixto et al., (2000) was applied with

109 some modifications; Control-samples (without UAE treatment) and UAE-samples were

110 submitted to an in vitro digestion as is shown in Fig. 1. Step 1: Control-samples and UAE-

111 samples (300 mg, peels and paste) were incubated with pepsin (0.2 ml of 300 mg/ml HCl–

112 KCl 0.2 M buffer, pH 1.5, 40°C, 1 hr, P7000, Sigma-Aldrich), after which samples were

113 considered to be the gastric fraction (GF). Step 2: Afterwards, samples were submitted to a

114 hydrolysis with pancreatin (1 ml of 5 mg/ml phosphate buffer 0.1 M, pH 7.5, 37°C, 6 hr,

115 Sigma P1750) and α-amylase (1 ml of 120 mg/ml tris-maleate buffer 0.1 M; pH 6.9, 37°C,

116 16 hr, Sigma A-3176); after this period, this hydrolyzed samples were considered to be the

117 intestinal fraction (IF). Step 3: After the enzymatic hydrolysis, the samples were

118 centrifuged (15 min, 25°C, 4000 rpm) to separate the soluble and insoluble indigestible

119 fractions. Step 4: The supernatants were removed and the residues were washed twice with

5
120 distilled water (5 ml), and all supernatants were combined. The supernatants were dialyzed

121 against water in a dialysis membrane (48 hr, 12–14 KDa, D9652, Sigma Aldrich). Step 5:

122 The residues were used to evaluate the carotenoids associated with the insoluble

123 indigestible fraction (IIF). Step 6: After the dialysis time, the carotenoids associated with

124 the soluble indigestible fraction (SIF) were evaluated. The carotenoids associated with the

125 IIF and SIF correspond to the non-bioaccessible carotenoids. Carotenoid content in the

126 different fractions: GF, IF, SIF, and IIF were evaluated as described in the following

127 section. The in vitro percentage of the bioaccessibility (%BA) of carotenoids was

128 determined using the following Eq (1)

(𝐼𝐹 ‒ 𝑆𝐼𝐹)
129 %𝐵𝐴 = (𝐼𝐹 + 𝐼𝐼𝐹)
(1)

130 IF: Carotenoids released in the intestinal fraction; SIF: Carotenoids associated with the
131 soluble indigestible fraction; IIF: carotenoids associated with the insoluble indigestible
132 fraction.
133 The Non-bioaccessible percentage of carotenoids (%NBA) was calculated using the Eq (2):

134 %𝑁𝐵𝐴 = 100 ‒ %𝐵𝐴 (2)

135 2.5 Carotenoid content of the in vitro digested fractions

136 Carotenoids were extracted from the GF, IF, SIF, and IIF following the methodology

137 described by De Ancos et al. (2000) with slightly modifications. Digested fractions were

138 dissolved with tetrahydrofuran (10 ml), each fraction was dried, and the residue was

139 dissolved in 1 ml of acetonitrile (HPLC grade). The extracted fraction was filtered through

140 a 0.45 µm membrane filter (MFTM Membrane filters, Millipore, California USA). Samples

141 were analyzed and quantified on a Dionex HPLC-PAD system (Dionex®, ICS-5000, USA),

142 using a C30 normal phase column (Thermo Scientific®, 5 µm particle size Vydac 201TP54,

143 3.0 mm in diameter, Walttham, MA, USA) and a C30 guard column (20×4.6 mm; Thermo

6
144 Scientific®, Wilmington, NC, USA) at room temperature (25°C). The results were analyzed

145 by Chromeleon Management software. β-cryptoxanthin (βCr), β-carotene (βC), and lutein

146 (Lut) were identified by comparing the retention time (tR) and the absorption spectra with

147 commercial standards. Standard solutions of Lut (0.39 – 22 µM), βCr (0.16 – 18 µM), and

148 βC (11 – 372 µM) were prepared in acetonitrile (HPLC grade, Fisher Scientific) and were

149 divided into five calibration points. Standards solutions and mobile phases were degassed

150 and filtered through a 0.45 µm membrane filter (MFTM Membrane filters, Millipore). All of

151 the extractions were carried out in triplicate. The final content of βC, βCr and Lut was

152 expressed as mg/g dry weight (DW).

153 2.6 In vitro kinetics of carotenoids release in mango by-products

154 The in vitro kinetics of βC, βCr, and Lut release from mango ‘Ataulfo’ BPs was determined

155 in the mango by-products treated with UAE and Control samples according to the in vitro

156 digestion method of Blancas-Benítez et al., (2015) with slight modifications. Enzymatic-

157 treatment was carried out in a glass vessel with 200 ml of phosphate buffer (0.05 M, pH

158 6.9), previously stabilized at 37°C. The samples were incubated for 3 hr with continuous

159 stirring. At 30 min intervals, 5 ml of the external liquid containing the dialyzed compounds

160 was collected, and the carotenoids were extracted and analyzed from aliquots of each

161 digestion at each time interval. The βC, βCr and Lut contents were expressed as mg/g dry

162 weight (DW).

163 2.7 Kinetic parameters of carotenoids released during the in vitro digestion

164 The initial rates (vo) of βC, βCr, and Lut release were determined according to the Eq (3):

(𝐶1 ‒ 𝐶0)
165 𝑣𝑜 = (𝑡1 ‒ 𝑡0)
(3)

7
166 where vo is the rate of carotenoid released in a specific time during the in vitro digestion

167 (mg/min), C1-C0 is the difference of concentrations between the concentration at a specific

168 time and the initial concentration (mg/g DW), and t1-t0 is the difference in time between a

169 specific time and the initial time (min).

170 The final rate (Vf) and the kinetic constant (k) of βC, βCr and Lut release were determined

171 in Eq (4) and (5):

△𝐶
172 𝑉𝑓 = ∑( △ 𝑡 ) (4)

𝑉𝑓
173 𝑘 = [ 𝐶] (5)

174 where △C is the difference in concentration between the concentration at a specific time

175 and the initial concentration, △t is the difference in time between a specific time and the

176 initial time, Vf is the final rate of the carotenoid released during the in vitro digestion.

177 The kinetics of βC, βCr, and Lut content were described by fitting a zero order or a first-

178 order kinetic model to the experimental data in Eq (6) and (7)

179 𝐴 = 𝐴0 ‒ 𝑘𝐶 (6)

𝐴 ‒ 𝑘𝐶
180 𝐴0
= 𝑒 (7)

181 where A is the parameter to be estimated, the subscript 0 indicates the initial value of the

182 parameter, t is each time, and k is the kinetic constant at concentration C. For the parameter

183 estimation, the individual measurements of the concentrations were used instead of the

184 mean values of duplicate experiments, thereby taking into account variability among

185 samples.

186 2.8 Statistical analysis

8
187 All analyses were performed at least in triplicate. The Statistica Version 10 software was

188 used for statistical analysis. The results were analyzed statistically using a one-way

189 ANOVA test, and differences between samples and treatments were determined using

190 Tukey's HSD multiple rank-test (p < 0.05).

191

192 3. Results and Discussion

193 3.1 Effect of UAE on the release of carotenoids in GF and IF of mango by-products

194 Table 1 shows the results of the carotenoid content in the Control and UAE samples for the

195 GF and IF. When UAE is applied, there is a positive effect on the βCr content, which

196 showed a significant difference (p<0.05) in all samples. The UAE-peel had a significantly

197 higher βCr content (3.59 ± 0.43 mg/g DW) than that of the Control-peel (0.66 ± 0.03 mg/g

198 DW). These results may be due to the cavitation influence on the structure of the cell wall

199 that released the βCr (Anesse et al., 2015; Hadiyanto et al., 2015). In this way, the βCr

200 content depends on its lipophilic form, which links with other lipids or carotenoids (Lut, α-

201 and β-carotene) that facilitate its release (Nwachukwu et al. 2016; Cervantes-Paz et al.

202 2016). Lut was not quantified in the peels but was quantified in both pastes in the GF.

203 These results differ because of the difference in the DF content between the peel (41.34%)

204 and paste (14.97%) (Blancas-Benitez et al., 2015). Additionally, the presence of divalent

205 metals can affect the release of carotenoids (Cervantes Paz et al. 2016; Corte-Real et al.

206 2016). However, the Lut content was higher in the Control-paste than in the UAE-paste.

207 These results may be observed because the Lut content decreases when the extraction time

208 is longer than 15 min (Altemimi et al., 2015). Conversely, the βC content presented

209 significant differences (p<0.05) between all samples. However, the UAE-paste had a lower

210 βC content than the Control-paste. These results could be explained by the fact that βC

9
211 exists in a di-esterified form, which can retard its release (Bunea et al., 2014). These results

212 suggest that the GF is a critical factor for the subsequent absorption of carotenoids in the

213 small intestine, since their stability will depend on their polarity, structure, and interactions

214 with other compounds of the food matrix (Altemimi et al., 2015; Estévez-Santiago et al.,

215 2016).

216 In the IF, the βCr and Lut content in the peels (Control and UAE) showed significant

217 differences (p<0.05), but significant differences were not observed for the paste samples.

218 The Lut content was higher than that reported in fruits (orange, grape, and mandarin

219 orange) analyzed by Mamatha and Baskaran (2011). These results can be attributed to

220 cavitation, since it promotes the hydrolytic activity of lipase and α-amylase which release

221 carotenoids partially bond to other compounds (Kiokias et al., 2016; Cervantes-Paz et al.,

222 2016). During the hydrolysis, other carotenoids can be released (α-cryptoxanthin and

223 zeinoxanthin) with structural changes, leading to the formation of Lut (Reboul 2013). The

224 βC content in UAE-peel was higher than that in the Control-peel, and the same trend was

225 observed in the pastes. These results suggest that the βC released with UAE is more

226 susceptible to be solubilized by micelles and, consequently, can be absorbed in the small

227 intestine (Reboul 2013).

228

229 3.2 Effect of UAE on non-released carotenoids in SIF and IIF of mango by-products

230 During the digestion process, certain carotenoids were not released from the food-matrix,

231 either by intrinsic (e.g., digestion process, enzymatic activity, and pH) or extrinsic factors

232 (e.g., food matrix and interaction between components) (Reboul 2013). Therefore, the βCr,

233 βC, and Lut contents were quantified in the SIF and IIF (Table 2). The SIF showed that for

234 βCr and Lut, the contents were significantly different (p<0.05) in all studied samples, but

10
235 the βC content was only significantly different (p<0.05) in the peel samples and not in the

236 paste samples. These results could be observed because the UAE exerts an effect on the

237 structure of βC (Hervert-Hernández et al., 2010).

238 The βCr content SIF in Control-samples was lower than in the UAE-samples. These results

239 were similar to the βCr content in different varieties of chili peppers (Goñi et al., 2006).

240 However, a significant difference (p<0.05) was found between paste samples (Control and

241 UAE). Nevertheless, these results were higher than the values reported by Mamatha and

242 Baskaran (2011). However, the Lut content in Control samples was higher than in the UAE

243 samples. The βC content in the Control peel was lower than in the UAE-peel. Most likely,

244 the type of carbohydrate present in the SIF, such as pectin or hemicellulose, has a negative

245 effect on the release of these carotenoids (Burri et al., 2016). However, the structure of the

246 DF can change by thermal processes or during UAE, linking the carotenoids at its external

247 structure (Anesse et al., 2015). Table 2 shows the βCr, βC, and Lut contents in the IIF. The

248 βCr content was significantly different (p<0.05) between the samples and treatments. Both

249 of these results are similar to those reported by Li et al., (2013). However, Lut was not

250 detected in the peel in both samples (Control and UAE). Therefore, the βC content did not

251 show significant differences (p>0.05) between the samples. Carotenoids linked to the IIF

252 could have interactions with lignin or some non-starch polysaccharides (arabinoxylans,

253 arabinogalactan) (Rafiq et al., 2016). Likewise, the trapping of these compounds may have

254 positive effects in the large intestine, producing metabolites through microbiota

255 fermentation, which can help to prevent certain diseases in the colon (Sepp et al., 2011).

256 βC, Lut, βCr and their derived products associated with the SIF and IIF may act as

257 immunoregulators by reducing oxidative damage, inhibiting lipid peroxidation, increasing

11
258 natural killer cell cytotoxicity, and increasing the total number of T and B cells in the

259 peripheral blood (Sepp et al. 2011; Lin et al. 2016).

260

261 3.3 Effect of the UAE treatment on the bioaccessibility percentage (%BA) of carotenoids

262 The %BA from mango BPs is shown in Fig. 2; the %BA in UAE-peel improved by

263 46.93%, 35.21% and 32.62% for βCr, Lut, and βC, respectively, and the same behavior was

264 observed for the UAE-paste, with the treatment improving by 46.04%, 44.16%, and

265 44.01% the %BA for Lut, βCr, and βC, respectively. The %BA of βC was significantly

266 different between the Control-peel (p<0.05) and the UAE-peel; also, the Control-paste and

267 UAE-paste were significantly different (p<0.05). The %BA for βCr of the Control and

268 UAE samples were higher than the %BA reported for red chili (29.7%) and lower than that

269 of kiwi fruit (77%) and orange (97.8%) (Livny et al. 2003). The cell wall of mango paste

270 can suffer changes after the industrial process, making Lut and βC more bioaccessible

271 during digestion. Losdat et al. (2010) and Sepp et al. (2011) observed that βC is more

272 bioaccessible in carrot paste. However, the %BA of βC in the Control-peel was similar to

273 that reported by Serrano et al. (2005). The %BA of βC in the UAE-peel was higher

274 (32.62%) than that reported by Mamatha and Baskaran (2011) for mandarin (25%) and

275 lower than that of orange (35%). In the same sense, the %BA of βC in the Control-paste

276 and UAE-paste were higher than that found in European black nightshade (Solanum

277 americanum) and chaya (Cnidoscolus aconitifolius) (Hervert-Hernández et al. 2010). The

278 %BA of carotenoids differs due to the treatments, content and the type of food matrix

279 (Dhuique-Mayer et al. 2007). Certainly, the UAE treatment improved the %BA in both

280 samples studied.

12
281 Fig. 3 shows the percentage of non-bioaccessible (%NBA) carotenoids in the IIF. For both

282 Control samples, the %NBA showed significant differences (p<0.05) with the UAE

283 samples. However, the %NBA of βC was 79.48% (Control-peel) and 70.41% (Control-

284 paste), and 67.37% (UAE-peel) and 55.99% (UAE-paste). In this way, the viscosity

285 generated by the DF and the SIF interferes with the bile salts to decrease the incorporation

286 of carotenoids into micelles and their subsequent uptake into the enterocyte (Reboul 2013;

287 Li et al., 2013). However, when UAE is applied, these values were reduced due to the

288 cavitation which modifies the structure of the SIF and IIF by breaking ester bonds,

289 hydrogen bonds, and other bonds (Chemat et al., 2017), which leads to increased BA of

290 carotenoids and, in consequence, reduces the non-bioaccessible carotenoids. It is known

291 that cutin is a component of IIF, and this polymer, composed of di- and tri- hydroxy and

292 epoxy fatty acids (hydroxypalmitic acid, oleic acid and hydroxyoleic acid), is commonly

293 found linked to carotenoids (Arabani et al., 2015).

294

295 3.4 In vitro kinetics of the release of carotenoids

296 Table 3 shows in vitro kinetics of the release of βCr, Lut, and βC content from mango BPs

297 (up to 180 min), and certain carotenoids are released in the first 30 min. Therefore, the

298 transport of βCr, Lut and βC release can be due to four factors: the free and/or bonded

299 form, the content of carotenoids in each sample (peel and paste), the type of transport of the

300 carotenoids, and the effect of UAE on the structure and content of the carotenoids

301 (Hadiyanto et al. 2015). Likewise, the transport of Lut is governed by facilitated diffusion

302 when this carotenoid is free, esterified or in micelles, or in the presence of other carotenoids

303 (zeaxanthin, lycopene, astaxanthin, canthaxanthin) (Hadiyanto et al. 2015; Moorthy et al.

304 2015).

13
305 The release of βC can be affected by the properties of the soluble DF, as shown by previous

306 studies (von Lintig et al., 2005). Additionally, the high release of βCr and Lut by cavitation

307 can interfere with the release of the βC. Similarly, previous studies have demonstrated that

308 polar carotenoids (Lut, βCr, astaxanthin, capsanthin, and others) are more bioaccessible

309 than the non-polar carotenoids (βC) (Estévez-Santiago et al., 2016; Hadiyanto et al., 2015).

310

311 3.5 Kinetic parameters of carotenoids released during the in vitro digestion

312 The release rate of βCr, Lut, and βC in mango BPs are shown in Fig. 4. The release rate of

313 βCr and Lut was improved in the UAE samples. However, for βC, the release rate was

314 decreasing over time and was higher for the UAE-peel. The kinetic parameters in the BP

315 samples are shown in Table 4. The Vf of βCr and Lut in the Control-peel were higher than

316 in the Control-paste. The Vf represents the reactions of α-amylase to hydrolyze the

317 glycosidic bonds in order to release the carotenoids. The Vf of βCr and βC were higher in

318 the UAE-peel than the UAE-paste. However, the Vf of Lut was similar in both samples with

319 UAE. These results can be observed because the release rate is related to the concentration

320 of each carotenoid (Schweiggert et al., 2014).

321 Table 4 shows the k of carotenoids for both treatments. The k-values were similar between

322 each carotenoid for the treatments, which means that UAE did not affect the release

323 kinetics. The k-values represent the factors that influence the release of carotenoids, such as

324 the enzymatic activity (α-amylase) and the effect of the constant movement and porosity on

325 the dialysis tubing (Palafox-Carlos et al., 2011; Schweiggert et al., 2014). The model that

326 was a better fit to the release kinetics was a 2-parameter, non-linear regression. The

327 considerations regarding diffusion were admitted, assuming that 1) carotenoids were

328 uniformly distributed within the samples, 2) diffusivity remained constant throughout the

14
329 extraction process, 3) the digestion process was perfectly mixed upon the energy dissipated

330 by the ultrasonic waves, 4) resistance to mass transfer was negligible in the intestinal phase,

331 and 5) the transport of carotenoids (βCr, Lut and βC) from the solid particles into the liquid

332 phase occurred through diffusion. The effect of UAE on the release of carotenoids could be

333 due to the increase in solubility observed during the intestinal process (Anesse et al., 2015).

334

335 4. Conclusions

336 The use of an in vitro gastrointestinal model was useful to provide information on in vitro

337 bioaccessibility of carotenoids from mango by-products. The in vitro bioaccessibility of

338 carotenoids increased with the application of UAE. βCr was more bioaccessible in the

339 UAE-peel, and Lut and βC were more bioaccessible in both BP with UAE. Therefore, UAE

340 of BPs could be used as an emergent technology that is more effective in increasing the

341 bioaccessibility of carotenoids. In the same way, the release kinetics of carotenoids was

342 better with UAE, which caused the structure of the carotenoids to be more exposed for their

343 release. The UAE was demonstrated to be a good alternative method for the release of these

344 compounds.

345

346 CONFLICTS OF INTEREST

347 The authors declare that they have no conflicts of interest.

348 Acknowledgments

349 GMM thanks Consejo Nacional de Ciencia y Tecnología (CONACyT) for the scholarship

350 he received, number 241180.

351

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Fig. 1. Diagram of in vitro digestion of carotenoids from the food matrix and associated

with indigestible fraction: (1) carotenoids released by gastric fraction, (2) carotenoids

released by intestinal fraction, (3) separate supernatant by centrifugation, (4) dialysis

membrane, (5) carotenoids associated to insoluble indigestible fraction, (6) carotenoids

associated to soluble indigestible fraction.

Fig. 2. Percentage of bioaccessible of β-cryptoxanthin, lutein, and β-carotene of peel and

paste. Different lowercase letters indicated difference between samples and upper letters

indicated difference between treatments (p< 0.05).

Fig. 3. Percentage of non-bioaccessible of β-cryptoxanthin, lutein, and β-carotene of peel

and paste
Fig. 4. Rate release of β-cryptoxanthin (A), lutein (B) y β-carotene (C) in mango by-

products
 Table 1. Carotenoids β-cryptoxanthin (βCr), lutein (Lut), and β-carotene (βC) content in
the gastric fraction (GF) and intestinal fraction (IF) in control and ultrasound-assisted
extraction (UAE) of mango by-products.
Peel (mg/g DW) Paste (mg/g DW)
Control UAE Control UAE

βCr 0.66 ± 0.03D 3.59 ± 0.43A 0.73 ± 0.03C 0.82 ± 0.03B

GF Lut n.q. n.q. 5.90 ± 0.05A 1.31 ± 0.52B

βC 13.62 ± 0.03D 14.79 ± 0.05C 19.35 ± 0.32A 17.59 ± 1.36B

βCr 1.52 ± 0.07B 5.76 ± 0.11A 1.11 ± 0.06B 1.45 ± 0.02B

IF Lut 13.22 ± 0.13B 15.25 ± 0.60A 7.79 ± 0.60C 7.34 ± 0.14C

βC 27.47 ± 0.05B 48.63 ± 0.81A 22.71 ± 0.67B 26.19 ± 0.18B

1UAE: By-product treated with ultrasound-assisted (XET: 30 min, XSA: 30 %, XPC: 0.8).
Values are the means of three replicates ± standard deviation (SD) (n=3). Values with
different upper letters are significantly different (p < 0.05). n.q.: no quantified.
 Table 2. Carotenoids β-cryptoxanthin (βCr), lutein (Lut), and β-carotene (βC) content in
soluble indigestible fraction (SIF) and insoluble indigestible fraction (IIF) in control and
ultrasound assisted extraction (UAE) treatments in mango by-products.

Peel (mg/g DW) Paste (mg/g DW)

Control UAE Control UAE

βCr 0.42 ± 0.02C 2.09 ± 0.07A 0.12 ± 0.05D 0.58 ± 0.01B

SIF Lut 10.41 ± 0.38A 9.88 ± 1.20B 4.48 ± 0.26C 3.96 ± 0.22D

βC 21.31 ± 0.30B 32.02 ± 0.75A 15.32 ± 0.32C 13.77 ± 0.14C

βCr 1.77 ± 0.08B 2.06 ± 0.05A 1.72 ± 0.04B 0.52 ± 0.00C

IIF Lut n.d n.d. n.d. n.d.

βC 2.56 ± 0.12A 2.28 ± 0.36A 2.27 ± 0.93A 2.03 ± 0.66A

1UAE: By-product treated with ultrasound-assisted (XET: 30 min, XSA: 30 %, XPC: 0.8).
Values are the means of three replicates ± standard deviation (SD) (n=3). Values with
different upper letters are significantly different (p < 0.05). n.d.: no detected.
 Table 3. In vitro Kinetics release of β-cryptoxanthin (βCr), lutein (Lut), and β-carotene
(βC) content in control and ultrasound-assisted extraction (UAE) in mango by-products.

Peel (mg/g DW) Paste (mg/g DW)

Time (min) Control UAE Control UAE
βCr 30 n.d. n.d. n.d. n.d.
60 0.07 ± 0.001A n.d. n.d. n.d.
90 0.08 ± 0.003B 0.17 ± 0.005A n.d.D 0.01 ± 0.002C
120 0.02 ± 0.001D 0.23 ± 0.007A 0.08 ± 0.004B 0.04 ± 0.001C
150 0.21 ± 0.002A 0.26 ± 0.001A 0.19 ± 0.01B 0.12 ± 0.003C
180 0.23 ± 0.01B 0.37 ± 0.007A 0.21 ± 0.002B 0.17 ± 0.01C

Lut 30 n.d. n.d. 0.03 ± 0.003A n.d.

60 0.36 ± 0.02A 0.14 ± 0.01B 0.39 ± 0.01A 0.38 ± 0.03A
90 1.27 ± 0.04A 1.19 ± 0.03A 0.85 ± 0.02B 1.45 ± 0.07A
120 1.77 ± 0.07B 2.14 ± 0.04A 1.30 ± 0.01B 2.47 ± 0.12A
150 2.58 ± 0.03A 2.92 ± 0.01A 1.89 ± 0.04B 2.89 ± 0.02A
180 3.03 ± 0.04A 3.47 ± 0.08A 2.55 ± 0.02B 3.11 ± 0.05A

βC 30 1.05 ± 0.000A 1.30 ± 0.36A 1.08 ± 0.001A 1.05 ± 0.002A

60 1.10 ± 0.001A 1.64 ± 0.001A 1.12 ± 0.003A 1.14 ± 0.007A
90 1.14 ± 0.002A 1.70 ± 0.004A 1.14 ± 0.002A 1.23 ± 0.001A
120 1.20 ± 0.001A 1.87 ± 0.003A 1.21 ± 0.002A 1.30 ± 0.004A
150 1.27 ± 0.006A 1.98 ± 0.007A 1.27 ± 0.004A 1.38 ± 0.002A
180 1.31 ± 0.001B 2.07 ± 0.005A 1.31 ± 0.003B 1.44 ± 0.005B

1UAE: By-product treated with ultrasound-assisted (XET: 30 min, XSA: 30 %, XPC: 0.8).
Values are the means of three replicates ± standard deviation (SD) (n=3). Values with
different upper letters are significantly different (p < 0.05). n.d.: no detected.
 Table 4. Kinetic parameters of β-cryptoxanthin (βCr), lutein (Lut) and β-carotene (βC) in
mango by-products.

Kinetic
Peel Paste
parameters
Control UAE Control UAE
βCr
Vf 0.002 0.003 0.0009 0.001

k 0.013 0.013 0.014 0.014

Lut
Vf 0.069 0.072 0.053 0.079

k 0.008 0.009 0.006 0.009

βC
Vf 0.091 0.130 0.093 0.095

k 0.001 0.002 0.001 0.002

Vf: the final rate (mg/min); k: the kinetic constant.