Accepted Manuscript In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following simulated gastro-intestinal digestion Ana Cristina Silva de Lima, Denise Josino Soares, Larissa Morais Ribeiro da Silva, Raimundo Wilane de Figueiredo, Paulo Henrique Machado de Sousa, Eveline de Abreu Menezes PII: DOI: Reference:
S0308-8146(14)00526-3 http://dx.doi.org/10.1016/j.foodchem.2014.03.123 FOCH 15653
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
7 October 2013 20 March 2014 26 March 2014
Please cite this article as: de Lima, A.C.S., Soares, D.J., da Silva, L.M.R., de Figueiredo, R.W., de Sousa, P.H.M., de Abreu Menezes, E., In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following simulated gastro-intestinal digestion, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.03.123
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
1
In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole
2
cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following
3
simulated gastro-intestinal digestion
4 5
Running title: Bioaccessibility of cashew apple following simulated gastro-intestinal
6
digestion
7 8
Ana Cristina Silva de Limaa*, Denise Josino Soaresa, Larissa Morais Ribeiro da Silvaa,
9
Raimundo Wilane de Figueiredoa, Paulo Henrique Machado de Sousab, Eveline de Abreu
10
Menezesc
11
12
a
Departamento de Tecnologia de Alimentos/Universidade Federal do Ceará, Av. Mister Hull,
13
2977, Campus Universitário do Pici, Fortaleza, Ceara, Brazil 60356-000. E-mail:
14
[email protected];
15
[email protected]
16
b
[email protected];
[email protected];
Instituto de Cultura e Arte/Universidade Federal do Ceará, Av. Mister Hull, 2977, Campus
do
Pici,
Fortaleza,
Ceara,
Brazil.
60356-000.
E-mail:
17
Universitário
18
[email protected]
19
c Universidade Estadual do Paiuí, Campus Professor Antonio Geovanne de Sousa Piripiri.
20
Avenida Marechal Castelo Branco, 180 Petecas, Piripiri, Piaui, Brazil, 64260-000. E-mail:
21
[email protected]
22
23 24
* Corresponding author: E-mail: [email protected]; Fax: +55 85 33669752.
2
25
Abstract. Considering the lack of research studies about nutrients’ bioaccessibility in cashew
26
apple, in this study the whole cashew apple juice and the cashew apple fiber were submitted
27
to simulated in vitro gastrointestinal digestion. The samples were analyzed before and after
28
digestion and had their copper, iron, zinc, ascorbic acid, total extractable phenols and total
29
antioxidant activity assessed. As a result, for the whole cashew apple juice, the content of
30
copper and iron minerals bioaccessible fraction was higher than 10% and for zinc this level
31
was lower than 5%. Regarding the cashew apple fiber, the bioaccessible fraction for these
32
minerals was lower than 5%. The ascorbic acid, total extractable polyphenols and total
33
antioxidant activity bioaccessible fraction for whole cashew apple juice showed
34
bioaccessibility percentages higher than 25%, while for the cashew apple fiber, bioaccessibles
35
levels were found to be around 15%.
36
Key-Words: ICP-OES, inorganic compounds, bioactive compounds, antioxidant activity.
37 38 39
3
40
In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole
41
cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following
42
simulated gastro-intestinal digestion
43 44
Running title: Bioaccessibility of cashew apple juice and cashew apple fiber compounds.
45 46
Abstract. Considering the lack of research studies about nutrients’ bioaccessibility in cashew
47
apple, in this study the whole cashew apple juice and the cashew apple fiber were submitted
48
to simulated in vitro gastrointestinal digestion. The samples were analyzed before and after
49
digestion and had their copper, iron, zinc, ascorbic acid, total extractable phenols and total
50
antioxidant activity assessed. As a result, for the whole cashew apple juice, the content of
51
copper and iron minerals bioaccessible fraction were 15% and 11.5% and for zinc this level
52
was 3.7%. Regarding the cashew apple fiber, the bioaccessible fraction for these minerals was
53
lower than 5%. The ascorbic acid, total extractable polyphenols and total antioxidant activity
54
bioaccessible fraction for whole cashew apple juice showed bioaccessibility percentages of
55
26.2%, 39% and 27%, respectively, while for the cashew apple fiber, low bioaccessibles
56
levels were found. The bioacessible percentage of zinc, ascorbic acid and total extractable
57
polyphenols were higher in cashew apple juice than cashew apple fiber,
58
Key-Words: ICP OES, minerals, bioactive compounds, antioxidant activity.
59 60
1. Introduction
61
The cashew tree belongs to the Anacardiaceae family, concentrated in the tropical
62
region of the globe and is widespread in several countries such as Brazil, India, Mozambique,
63
Tanzania, Kenya, Vietnam, Indonesia and Thailand (Mazetto, Lomonaco, & Mele, 2009). The
64
pseudo fruit of the cashew tree is known as cashew apple and has a similar structure to that of
4
65
a fibrous and juicy fruit. The cashew apple pulp is rich in ascorbic acid (Queiroz, Lopes,
66
Fialho, & Valente-Mesquita, 2011), phenolic compounds, minerals (Sivagurunathan,
67
Sivasankari, & Muthukkaruppan, 2010) and carotenoids, giving this fruit the title of
68
functional food.
69
Despite its high level of astringency, cashew apple has a good potential for
70
industrialization, due to its fleshy pulp, soft skin, high sugar and exotic flavor
71
(Sivagurunathan et al., 2010), and it is widely consumed in the form of juice, nectar, jam,
72
among others. During the processing of fruit products byproducts such as skins, seeds and
73
fibers are produced. The utilization of these byproducts can contribute to the improvement of
74
the environment, in view of the large volumes produced and disposed of in inappropriate
75
places, causing serious environmental problems (Sousa, Vieira, Silva, & Lima, 2011). The use
76
of cashew apple fiber for human consumption opens up new perspectives, as it is a natural
77
source of phenolic compounds and antioxidant activity (Broinizi et al., 2007), and also has
78
appreciable amounts of vitamin C (Uchoa, Costa, Maia, Silva, Carvalho, & Meira, 2008).
79
Several studies have focused on the functional compounds present in cashew apple
80
(Brito, Araújo, Lin, & Harnly, 2007; Queiroz et al., 2011), however, in terms of nutrition it is
81
not enough merely to determine the total content of nutrients, it is necessary to know the
82
bioaccessibility, in other words, the amount of compound released from the matrix during
83
gastrointestinal digestion that becomes available for absorption in the intestine.
84
Studies about the bioaccessibility of nutrients in foods can be performed using in vivo
85
and/or in vitro methods. The combination of these methods can provide information that can
86
help in the interpretation of results. The in vitro method is applied to a system of simulated
87
gastrointestinal digestion using pepsin in the gastric phase and a mixture of pancreatin and
88
bile salts during the intestinal tract. The element diffused through a semipermeable membrane
5
89
in the intestinal phase is used as a measure of the element bioaccessibility (Kulkarni, Acharya,
90
Rajurkar, & Reddy, 2007).
91
In this context, the aim of this study was to determine the bioaccessibility of the
92
minerals copper, iron and zinc, of ascorbic acid, total phenolics and total antioxidant activity
93
of cashew apple juice and cashew apple fiber.
94
2. Material and Methods
95 96
2.1.
Chemicals
97
ABTS•+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), pancreatin, pepsin,
98
bile extract, Folin–Ciocalteau reagent, ascorbic acid and galic acid were purchased from
99
Sigma Aldrich (Saint Louis, USA). All the other reagents and chemicals of analytical grade
100
were purchased from local sources. Certified reference material SRM 1547 - Peach Leaves,
101
the National Institute of Standard Technology (NIST, Gaithersburg, MD, USA) was used to
102
assess the accuracy of methods for determination of analytes; stock patterns of Cu, Fe and Zn
103
1000 mg L-1 solutions. Titrisol (Merck, Darmstadt, Germany) were used to prepare reference
104
solutions.
105
2.2.
Cashew apple juice and cashew apple fiber
106
The experiment was performed with cashew apple juice and cashew apple fiber. Bottles
107
of cashew apple juice (500 mL) from three different batches belonging to a commercial brand,
108
were donated by the production company, located in Ceara/Brazil. For the extraction of
109
cashew apple fiber, whole fruits were used, with red and yellow color, hand-picked for their
110
quality attributes and washed by immersion in chlorinated water (200 ppm of active chlorine).
111
The cashew apples were peeled and the juice was separated from the fiber with the aid of a
112
domestic centrifuge. At the end of the process, the fibers which had been obtained were
113
packed in polyethylene bags, properly sealed in a vacuum and stored in a freezer (-18 ± 1°C)
114
until analysis.
115
2.3.
Mineral analysis
6
116
The digestion was performed with the aid of a microwave oven cavity (Model
117
Multiwave 3000) with a temperature sensor and internal and external pressure. Quartz jars
118
were used for digestion of the samples, adapting procedures described by Menezes (2010).
119
For the microwave digestion of the cashew apple samples, 0.5 mL of the juice was used,
120
which had been digested with the diluted oxidant mixture (1 mL of H2O2 and 2 mL of HNO3).
121
Table 1 describes the heating program used during microwave digestion of cashew apple
122
juice.
123
The quantification of copper, iron and zinc was performed by using an external standard
124
curve prepared with stock standard solutions of Cu, Fe e Zn 1000 mg L-1 Titrisol (Merck,
125
Darmstadt, Germany) as a reference. Certified reference material SRM 1547 - Peach Leaves,
126
the National Institute of Standard Technology (NIST, Gaithersburg, MD, USA) was used to
127
assess the accuracy of methods for determination of analytes.
128
After the complete oxidation of the organic matter, the samples were diluted to 20 mL
129
with deionized water and the copper, iron and zinc were measured by Inductively Coupled
130
Plasma Optical Emission Spectrometry (ICP OES) (Table 2).
131 132 133 134 135 136 137 138
For the microwave digestion of cashew apple fiber crushing of the samples was performed followed by drying in an oven at 60°C/24 hours and then maceration until a powder was obtained. Two hundred milligrams of the sample were digested with the diluted oxidant mixture (1 mL of H2O2, 1 mL of HNO3 and 1 mL of Milli Q water). After the complete oxidation of the organic matter, the samples were diluted to 10 mL with deionized water and the copper, iron and zinc were measured by ICP OES in the same way as described for the cashew apple juice using the heating program for microwave digestion as described in Table 1.
139
2.4.
Simulated in vitro gastrointestinal digestion
140
The digestions were performed with simulated gastric fluid and simulated intestinal
141
fluid, both prepared according to procedures implemented by Moura and Canniatti-Brazaca
142
(2006). The simulated gastrointestinal digestion was performed with pepsin solubilized with
143
0.1 mol L-1 HCl during the gastric phase and pancreatin-bile salts solubilized with 0.1 mol L-1
7
144
NaHCO3 in the intestinal phase. Twenty grams of each sample was added to 100 mL of 0.01
145
mol L-1 HCl and adjusted to pH 2 with 2 mol L-1 HCl solution. After the pH adjustment, 3.2
146
mL of pepsin was added, and the sample was stirred in a thermostat at 37°C/2 hours to
147
simulate the digestion of the food in the stomach. After that, titration with 0.5 mol L-1 NaOH
148
was performed until pH 7.5 to simulate the pH found in the intestine of an individual. Dialysis
149
was performed for two hours in dialysis membranes (33 x 21 mm, molecular weight: 12.000-
150
16.000, porosity: 25 Angstrons – INLAB, Brazil) containing 0.1 mol L-1 NaHCO3 equivalent
151
to titratable acidity. After the pH adjustment, the dialysis membranes were added and stirred
152
in a water bath thermostatted at 37°C/30 minutes, then 5.0 mL of pancreatin solution and bile
153
salts were added and stirred in a bath at 37°C/2 hours. This step simulates the digestion of
154
food in the intestine. At the end of this step the contents of the membrane (dialysate) were
155
removed and samples were stored at -20°C until the time of analysis.
156
2.5.
Determination of bioacessible levels of cooper, iron and zinc
157
The dialyzed samples obtained from the in vitro simulated gastrointestinal digestion
158
were analyzed by ICP OES according to the conditions mentioned in Table 2. The
159
bioaccessible percentage was calculated according to the equation described by Menezes
160
(2010) (Equation 1).
161 162
% Bioaccessible = (A x 25 mL/B x C) x 100
Equation (1)
163 164
A - element content of the dialysable mineral fraction (mg), B - value of the total mineral
165
(iron, copper or zinc) content of the sample (mg); C - initial weight of sample (g).
166 167
2.6.
Ascorbic acid level determination
8
168
The level of ascorbic acid was determined according to Scherer, Rybka and Godoy
169
(2008). Analyses were conducted in HPLC Shimadzu, controlled by LC Solution Software,
170
using a manual injector with a fixed volume of 20 µL, pump model LC-20DA, performed at
171
25°C adjusted by CTO-20A oven and UV-VIS detector model SPD-20A. The Nova Pack C18
172
(CLC-ODS, 3µm, 4.6 mm x 25 cm) column was used. The injections were performed in
173
triplicate. The mobile phase used was an aqueous solution of 0.01 M KH2PO4, with pH
174
adjusted to 2.6 with phosphoric acid at a flow rate of 0.5 mL min-1. The quantification was
175
performed by using an external standard curve with seven points prepared with ascorbic acid
176
(Sigma Aldrich, Saint Louis, USA) as a reference. All samples and the mobile phase were
177
filtered in a 0.45 µm membrane (Millipore). For the determination of ascorbic acid before the
178
simulated gastrointestinal digestion in vitro, cashew apple juice and cashew apple fiber were
179
diluted with the mobile phase (1/9, v/v, aqueous solution of 0.01 M KH2PO4), filtered and
180
injected in the chromatograph with a run time of 10 minutes. For the determination of
181
ascorbic acid after in vitro simulated gastrointestinal digestion of cashew apple juice and
182
cashew apple fiber the dialysate was removed, filtered and injected into the chromatograph
183
with a run time of 10 minutes under the same conditions described above. The identification
184
of ascorbic acid in the samples was performed by comparing the retention times obtained for
185
the standard (L-ascorbic acid), and co-injection of samples with the standard solution. The
186
bioaccessible percentage was calculated according to Briones-Labarca, Venegas-Cubillos,
187
Ortiz-Portilla, Chacana-Ojeda, and Maureira (2011) (Equation 2).
188 189
% Bioaccessible = 100 x (D/E)
Equation (2)
190 191
D - ascorbic acid dialyzable content (mg 100 g-1), E - ascorbic acid content of the sample (mg
192
100 g-1).
9
193
2.7.
Total extractable polyphenol level determination
194
The total extractable polyphenols were determined by the Folin-Ciocalteu method,
195
using a standard curve prepared with galic acid (Sigma Aldrich, Saint Louis, USA) as a
196
reference, according to the methodology described by Larrauri, Rupérez, and Saura-Calixto
197
(1997). The determination of total extractable polyphenols before in vitro simulated
198
gastrointestinal digestion of the cashew apple juice and cashew apple fiber was performed
199
according to the methodology described by Larrauri et al. (1997), by reading the extracts in a
200
spectrophotometer (Shimadzu, model UV-1800) at 700 nm. For the determination of total
201
extractable polyphenols after simulated gastrointestinal digestion in vitro the dialysate was
202
analyzed using the same methodology described for the determination of total extractable
203
polyphenols before gastrointestinal digestion. The results were expressed in mg of galic acid
204
equivalent (GAE) 100g-1. The bioaccessible percentage was calculated according to Briones-
205
Labarca et al. (2011) (Equation 3).
206 207
% Bioaccessible = 100 x (F/G)
Equation (3)
208 209
F – Total extractable polyphenol compounds dialyzable (mg GAE 100 g-1), G - total
210
extractable polyphenol content of the sample (mg GAE 100 g-1).
211 212
2.8.
Antioxidant activity
213
The antioxidant activity was determined by the ABTS•+ method, as described by
214
Rufino, Alves, Brito, Pérez-Jiménez, Saura-Calixto, and Mancini-Filho (2010). The extract
215
used for this analysis was the same as that used for the determination of total extractable
216
polyphenols. The readings for the determination of antioxidant activity before and after in
217
vitro digestion were carried out in a spectrophotometer (Shimadzu model UV-1800) to 734
10
218
nm, and the quantification of the antioxidant activity before digestion was performed on the
219
extract prepared with the cashew apple juice and the cashew apple fiber and the quantification
220
after digestion was performed in the dialysate. The quantification was performed by using an
221
external standard curve prepared with Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-
222
carboxylic acid) (Sigma Aldrich, Saint Louis, USA) as a reference. The results were
223
expressed as equivalent antioxidant Trolox® (TEAC) in µM g-1. The bioaccessible percentage
224
was calculated according to Briones-Labarca et al. (2011) (Equation 4).
225 226
% Bioaccessible= 100 x (H/I)
Equation (4)
227 228
H - dialysable antioxidant activity (µM g-1), I - antioxidant activity of the sample (µM g-1.).
229 230
2.9.
Statistical analysis
231
The experiment was conducted according to a completely randomized design with three
232
replications of the experiments. The results were statistically evaluated by variance analysis.
233
As evidenced the significant by the F test, the treatments were compared by Tukey test at 5%
234
probability.
235
3. Results and Discussion
236 237
The content of the minerals, ascorbic acid, total extractable polyphenols and antioxidant
238
activity of cashew apple juice and cashew apple fiber decreased during gastrointestinal
239
digestion, occurring a significant difference (p
S0308-8146(14)00526-3 http://dx.doi.org/10.1016/j.foodchem.2014.03.123 FOCH 15653
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
7 October 2013 20 March 2014 26 March 2014
Please cite this article as: de Lima, A.C.S., Soares, D.J., da Silva, L.M.R., de Figueiredo, R.W., de Sousa, P.H.M., de Abreu Menezes, E., In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following simulated gastro-intestinal digestion, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.03.123
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
1
In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole
2
cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following
3
simulated gastro-intestinal digestion
4 5
Running title: Bioaccessibility of cashew apple following simulated gastro-intestinal
6
digestion
7 8
Ana Cristina Silva de Limaa*, Denise Josino Soaresa, Larissa Morais Ribeiro da Silvaa,
9
Raimundo Wilane de Figueiredoa, Paulo Henrique Machado de Sousab, Eveline de Abreu
10
Menezesc
11
12
a
Departamento de Tecnologia de Alimentos/Universidade Federal do Ceará, Av. Mister Hull,
13
2977, Campus Universitário do Pici, Fortaleza, Ceara, Brazil 60356-000. E-mail:
14
[email protected];
15
[email protected]
16
b
[email protected];
[email protected];
Instituto de Cultura e Arte/Universidade Federal do Ceará, Av. Mister Hull, 2977, Campus
do
Pici,
Fortaleza,
Ceara,
Brazil.
60356-000.
E-mail:
17
Universitário
18
[email protected]
19
c Universidade Estadual do Paiuí, Campus Professor Antonio Geovanne de Sousa Piripiri.
20
Avenida Marechal Castelo Branco, 180 Petecas, Piripiri, Piaui, Brazil, 64260-000. E-mail:
21
[email protected]
22
23 24
* Corresponding author: E-mail: [email protected]; Fax: +55 85 33669752.
2
25
Abstract. Considering the lack of research studies about nutrients’ bioaccessibility in cashew
26
apple, in this study the whole cashew apple juice and the cashew apple fiber were submitted
27
to simulated in vitro gastrointestinal digestion. The samples were analyzed before and after
28
digestion and had their copper, iron, zinc, ascorbic acid, total extractable phenols and total
29
antioxidant activity assessed. As a result, for the whole cashew apple juice, the content of
30
copper and iron minerals bioaccessible fraction was higher than 10% and for zinc this level
31
was lower than 5%. Regarding the cashew apple fiber, the bioaccessible fraction for these
32
minerals was lower than 5%. The ascorbic acid, total extractable polyphenols and total
33
antioxidant activity bioaccessible fraction for whole cashew apple juice showed
34
bioaccessibility percentages higher than 25%, while for the cashew apple fiber, bioaccessibles
35
levels were found to be around 15%.
36
Key-Words: ICP-OES, inorganic compounds, bioactive compounds, antioxidant activity.
37 38 39
3
40
In vitro bioaccessibility of copper, iron, zinc and antioxidant compounds of whole
41
cashew apple juice and cashew apple fiber (Anacardium occidentale L.) following
42
simulated gastro-intestinal digestion
43 44
Running title: Bioaccessibility of cashew apple juice and cashew apple fiber compounds.
45 46
Abstract. Considering the lack of research studies about nutrients’ bioaccessibility in cashew
47
apple, in this study the whole cashew apple juice and the cashew apple fiber were submitted
48
to simulated in vitro gastrointestinal digestion. The samples were analyzed before and after
49
digestion and had their copper, iron, zinc, ascorbic acid, total extractable phenols and total
50
antioxidant activity assessed. As a result, for the whole cashew apple juice, the content of
51
copper and iron minerals bioaccessible fraction were 15% and 11.5% and for zinc this level
52
was 3.7%. Regarding the cashew apple fiber, the bioaccessible fraction for these minerals was
53
lower than 5%. The ascorbic acid, total extractable polyphenols and total antioxidant activity
54
bioaccessible fraction for whole cashew apple juice showed bioaccessibility percentages of
55
26.2%, 39% and 27%, respectively, while for the cashew apple fiber, low bioaccessibles
56
levels were found. The bioacessible percentage of zinc, ascorbic acid and total extractable
57
polyphenols were higher in cashew apple juice than cashew apple fiber,
58
Key-Words: ICP OES, minerals, bioactive compounds, antioxidant activity.
59 60
1. Introduction
61
The cashew tree belongs to the Anacardiaceae family, concentrated in the tropical
62
region of the globe and is widespread in several countries such as Brazil, India, Mozambique,
63
Tanzania, Kenya, Vietnam, Indonesia and Thailand (Mazetto, Lomonaco, & Mele, 2009). The
64
pseudo fruit of the cashew tree is known as cashew apple and has a similar structure to that of
4
65
a fibrous and juicy fruit. The cashew apple pulp is rich in ascorbic acid (Queiroz, Lopes,
66
Fialho, & Valente-Mesquita, 2011), phenolic compounds, minerals (Sivagurunathan,
67
Sivasankari, & Muthukkaruppan, 2010) and carotenoids, giving this fruit the title of
68
functional food.
69
Despite its high level of astringency, cashew apple has a good potential for
70
industrialization, due to its fleshy pulp, soft skin, high sugar and exotic flavor
71
(Sivagurunathan et al., 2010), and it is widely consumed in the form of juice, nectar, jam,
72
among others. During the processing of fruit products byproducts such as skins, seeds and
73
fibers are produced. The utilization of these byproducts can contribute to the improvement of
74
the environment, in view of the large volumes produced and disposed of in inappropriate
75
places, causing serious environmental problems (Sousa, Vieira, Silva, & Lima, 2011). The use
76
of cashew apple fiber for human consumption opens up new perspectives, as it is a natural
77
source of phenolic compounds and antioxidant activity (Broinizi et al., 2007), and also has
78
appreciable amounts of vitamin C (Uchoa, Costa, Maia, Silva, Carvalho, & Meira, 2008).
79
Several studies have focused on the functional compounds present in cashew apple
80
(Brito, Araújo, Lin, & Harnly, 2007; Queiroz et al., 2011), however, in terms of nutrition it is
81
not enough merely to determine the total content of nutrients, it is necessary to know the
82
bioaccessibility, in other words, the amount of compound released from the matrix during
83
gastrointestinal digestion that becomes available for absorption in the intestine.
84
Studies about the bioaccessibility of nutrients in foods can be performed using in vivo
85
and/or in vitro methods. The combination of these methods can provide information that can
86
help in the interpretation of results. The in vitro method is applied to a system of simulated
87
gastrointestinal digestion using pepsin in the gastric phase and a mixture of pancreatin and
88
bile salts during the intestinal tract. The element diffused through a semipermeable membrane
5
89
in the intestinal phase is used as a measure of the element bioaccessibility (Kulkarni, Acharya,
90
Rajurkar, & Reddy, 2007).
91
In this context, the aim of this study was to determine the bioaccessibility of the
92
minerals copper, iron and zinc, of ascorbic acid, total phenolics and total antioxidant activity
93
of cashew apple juice and cashew apple fiber.
94
2. Material and Methods
95 96
2.1.
Chemicals
97
ABTS•+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), pancreatin, pepsin,
98
bile extract, Folin–Ciocalteau reagent, ascorbic acid and galic acid were purchased from
99
Sigma Aldrich (Saint Louis, USA). All the other reagents and chemicals of analytical grade
100
were purchased from local sources. Certified reference material SRM 1547 - Peach Leaves,
101
the National Institute of Standard Technology (NIST, Gaithersburg, MD, USA) was used to
102
assess the accuracy of methods for determination of analytes; stock patterns of Cu, Fe and Zn
103
1000 mg L-1 solutions. Titrisol (Merck, Darmstadt, Germany) were used to prepare reference
104
solutions.
105
2.2.
Cashew apple juice and cashew apple fiber
106
The experiment was performed with cashew apple juice and cashew apple fiber. Bottles
107
of cashew apple juice (500 mL) from three different batches belonging to a commercial brand,
108
were donated by the production company, located in Ceara/Brazil. For the extraction of
109
cashew apple fiber, whole fruits were used, with red and yellow color, hand-picked for their
110
quality attributes and washed by immersion in chlorinated water (200 ppm of active chlorine).
111
The cashew apples were peeled and the juice was separated from the fiber with the aid of a
112
domestic centrifuge. At the end of the process, the fibers which had been obtained were
113
packed in polyethylene bags, properly sealed in a vacuum and stored in a freezer (-18 ± 1°C)
114
until analysis.
115
2.3.
Mineral analysis
6
116
The digestion was performed with the aid of a microwave oven cavity (Model
117
Multiwave 3000) with a temperature sensor and internal and external pressure. Quartz jars
118
were used for digestion of the samples, adapting procedures described by Menezes (2010).
119
For the microwave digestion of the cashew apple samples, 0.5 mL of the juice was used,
120
which had been digested with the diluted oxidant mixture (1 mL of H2O2 and 2 mL of HNO3).
121
Table 1 describes the heating program used during microwave digestion of cashew apple
122
juice.
123
The quantification of copper, iron and zinc was performed by using an external standard
124
curve prepared with stock standard solutions of Cu, Fe e Zn 1000 mg L-1 Titrisol (Merck,
125
Darmstadt, Germany) as a reference. Certified reference material SRM 1547 - Peach Leaves,
126
the National Institute of Standard Technology (NIST, Gaithersburg, MD, USA) was used to
127
assess the accuracy of methods for determination of analytes.
128
After the complete oxidation of the organic matter, the samples were diluted to 20 mL
129
with deionized water and the copper, iron and zinc were measured by Inductively Coupled
130
Plasma Optical Emission Spectrometry (ICP OES) (Table 2).
131 132 133 134 135 136 137 138
For the microwave digestion of cashew apple fiber crushing of the samples was performed followed by drying in an oven at 60°C/24 hours and then maceration until a powder was obtained. Two hundred milligrams of the sample were digested with the diluted oxidant mixture (1 mL of H2O2, 1 mL of HNO3 and 1 mL of Milli Q water). After the complete oxidation of the organic matter, the samples were diluted to 10 mL with deionized water and the copper, iron and zinc were measured by ICP OES in the same way as described for the cashew apple juice using the heating program for microwave digestion as described in Table 1.
139
2.4.
Simulated in vitro gastrointestinal digestion
140
The digestions were performed with simulated gastric fluid and simulated intestinal
141
fluid, both prepared according to procedures implemented by Moura and Canniatti-Brazaca
142
(2006). The simulated gastrointestinal digestion was performed with pepsin solubilized with
143
0.1 mol L-1 HCl during the gastric phase and pancreatin-bile salts solubilized with 0.1 mol L-1
7
144
NaHCO3 in the intestinal phase. Twenty grams of each sample was added to 100 mL of 0.01
145
mol L-1 HCl and adjusted to pH 2 with 2 mol L-1 HCl solution. After the pH adjustment, 3.2
146
mL of pepsin was added, and the sample was stirred in a thermostat at 37°C/2 hours to
147
simulate the digestion of the food in the stomach. After that, titration with 0.5 mol L-1 NaOH
148
was performed until pH 7.5 to simulate the pH found in the intestine of an individual. Dialysis
149
was performed for two hours in dialysis membranes (33 x 21 mm, molecular weight: 12.000-
150
16.000, porosity: 25 Angstrons – INLAB, Brazil) containing 0.1 mol L-1 NaHCO3 equivalent
151
to titratable acidity. After the pH adjustment, the dialysis membranes were added and stirred
152
in a water bath thermostatted at 37°C/30 minutes, then 5.0 mL of pancreatin solution and bile
153
salts were added and stirred in a bath at 37°C/2 hours. This step simulates the digestion of
154
food in the intestine. At the end of this step the contents of the membrane (dialysate) were
155
removed and samples were stored at -20°C until the time of analysis.
156
2.5.
Determination of bioacessible levels of cooper, iron and zinc
157
The dialyzed samples obtained from the in vitro simulated gastrointestinal digestion
158
were analyzed by ICP OES according to the conditions mentioned in Table 2. The
159
bioaccessible percentage was calculated according to the equation described by Menezes
160
(2010) (Equation 1).
161 162
% Bioaccessible = (A x 25 mL/B x C) x 100
Equation (1)
163 164
A - element content of the dialysable mineral fraction (mg), B - value of the total mineral
165
(iron, copper or zinc) content of the sample (mg); C - initial weight of sample (g).
166 167
2.6.
Ascorbic acid level determination
8
168
The level of ascorbic acid was determined according to Scherer, Rybka and Godoy
169
(2008). Analyses were conducted in HPLC Shimadzu, controlled by LC Solution Software,
170
using a manual injector with a fixed volume of 20 µL, pump model LC-20DA, performed at
171
25°C adjusted by CTO-20A oven and UV-VIS detector model SPD-20A. The Nova Pack C18
172
(CLC-ODS, 3µm, 4.6 mm x 25 cm) column was used. The injections were performed in
173
triplicate. The mobile phase used was an aqueous solution of 0.01 M KH2PO4, with pH
174
adjusted to 2.6 with phosphoric acid at a flow rate of 0.5 mL min-1. The quantification was
175
performed by using an external standard curve with seven points prepared with ascorbic acid
176
(Sigma Aldrich, Saint Louis, USA) as a reference. All samples and the mobile phase were
177
filtered in a 0.45 µm membrane (Millipore). For the determination of ascorbic acid before the
178
simulated gastrointestinal digestion in vitro, cashew apple juice and cashew apple fiber were
179
diluted with the mobile phase (1/9, v/v, aqueous solution of 0.01 M KH2PO4), filtered and
180
injected in the chromatograph with a run time of 10 minutes. For the determination of
181
ascorbic acid after in vitro simulated gastrointestinal digestion of cashew apple juice and
182
cashew apple fiber the dialysate was removed, filtered and injected into the chromatograph
183
with a run time of 10 minutes under the same conditions described above. The identification
184
of ascorbic acid in the samples was performed by comparing the retention times obtained for
185
the standard (L-ascorbic acid), and co-injection of samples with the standard solution. The
186
bioaccessible percentage was calculated according to Briones-Labarca, Venegas-Cubillos,
187
Ortiz-Portilla, Chacana-Ojeda, and Maureira (2011) (Equation 2).
188 189
% Bioaccessible = 100 x (D/E)
Equation (2)
190 191
D - ascorbic acid dialyzable content (mg 100 g-1), E - ascorbic acid content of the sample (mg
192
100 g-1).
9
193
2.7.
Total extractable polyphenol level determination
194
The total extractable polyphenols were determined by the Folin-Ciocalteu method,
195
using a standard curve prepared with galic acid (Sigma Aldrich, Saint Louis, USA) as a
196
reference, according to the methodology described by Larrauri, Rupérez, and Saura-Calixto
197
(1997). The determination of total extractable polyphenols before in vitro simulated
198
gastrointestinal digestion of the cashew apple juice and cashew apple fiber was performed
199
according to the methodology described by Larrauri et al. (1997), by reading the extracts in a
200
spectrophotometer (Shimadzu, model UV-1800) at 700 nm. For the determination of total
201
extractable polyphenols after simulated gastrointestinal digestion in vitro the dialysate was
202
analyzed using the same methodology described for the determination of total extractable
203
polyphenols before gastrointestinal digestion. The results were expressed in mg of galic acid
204
equivalent (GAE) 100g-1. The bioaccessible percentage was calculated according to Briones-
205
Labarca et al. (2011) (Equation 3).
206 207
% Bioaccessible = 100 x (F/G)
Equation (3)
208 209
F – Total extractable polyphenol compounds dialyzable (mg GAE 100 g-1), G - total
210
extractable polyphenol content of the sample (mg GAE 100 g-1).
211 212
2.8.
Antioxidant activity
213
The antioxidant activity was determined by the ABTS•+ method, as described by
214
Rufino, Alves, Brito, Pérez-Jiménez, Saura-Calixto, and Mancini-Filho (2010). The extract
215
used for this analysis was the same as that used for the determination of total extractable
216
polyphenols. The readings for the determination of antioxidant activity before and after in
217
vitro digestion were carried out in a spectrophotometer (Shimadzu model UV-1800) to 734
10
218
nm, and the quantification of the antioxidant activity before digestion was performed on the
219
extract prepared with the cashew apple juice and the cashew apple fiber and the quantification
220
after digestion was performed in the dialysate. The quantification was performed by using an
221
external standard curve prepared with Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-
222
carboxylic acid) (Sigma Aldrich, Saint Louis, USA) as a reference. The results were
223
expressed as equivalent antioxidant Trolox® (TEAC) in µM g-1. The bioaccessible percentage
224
was calculated according to Briones-Labarca et al. (2011) (Equation 4).
225 226
% Bioaccessible= 100 x (H/I)
Equation (4)
227 228
H - dialysable antioxidant activity (µM g-1), I - antioxidant activity of the sample (µM g-1.).
229 230
2.9.
Statistical analysis
231
The experiment was conducted according to a completely randomized design with three
232
replications of the experiments. The results were statistically evaluated by variance analysis.
233
As evidenced the significant by the F test, the treatments were compared by Tukey test at 5%
234
probability.
235
3. Results and Discussion
236 237
The content of the minerals, ascorbic acid, total extractable polyphenols and antioxidant
238
activity of cashew apple juice and cashew apple fiber decreased during gastrointestinal
239
digestion, occurring a significant difference (p
