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xanthan gum hydrogels produced by extrusion and thermopressing
Journal Pre-proof Citric acid as crosslinking agent in starch/xanthan gum hydrogels produced by extrusion and thermopressing Bruno Matheus Simões, Caroline Cagnin, Fabio Yamashita, Juliana Bonametti Olivato, Patrícia Salomão Garcia, Suzana Mali de Oliveira, Maria Victória Eiras Grossmann PII:
S0023-6438(19)31292-7
DOI:
https://doi.org/10.1016/j.lwt.2019.108950
Reference:
YFSTL 108950
To appear in:
LWT - Food Science and Technology
Received Date: 29 July 2019 Revised Date:
21 November 2019
Accepted Date: 12 December 2019
Please cite this article as: Simões, B.M., Cagnin, C., Yamashita, F., Olivato, J.B., Garcia, Patrí.Salomã., de Oliveira, S.M., Eiras Grossmann, Maria.Victó., Citric acid as crosslinking agent in starch/xanthan gum hydrogels produced by extrusion and thermopressing, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2019.108950. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Bruno Matheus Simões – Hydrogels processing, analysis of hydrogels properties, data analysis and manuscript redaction.
Caroline Cagnin – Hydrogels processing and analysis of some hydrogels properties.
Fábio Yamashita – Manuscript redaction.
Juliana Bonametti Olivato – DMA analysis and data discussion.
Patrícia Salomão Garcia – Manuscript redaction.
Suzana Mali de Oliveira – Manuscript redaction and hydrogels processing.
Maria Victória Eiras Grossmann – Data analysis and manuscript redaction.
1
Citric acid as crosslinking agent in starch/xanthan gum hydrogels produced by
2
extrusion and thermopressing
3 4
Bruno Matheus Simõesa, Caroline Cagnina, Fabio Yamashitaa, Juliana Bonametti Olivatob,
5
Patrícia Salomão Garciac, Suzana Mali de Oliveirad and Maria Victória Eiras Grossmanna*.
6 7
a
8
de Londrina, 86051-980 Londrina, PR, Brasil.
9
b
Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Estadual
Departamento de Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Câmpus Uvaranas,
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84030-900 Ponta Grossa, PR, Brasil.
11
c
12
Câmpus Apucarana, 86812-460 Apucarana, PR, Brasil.
13
d
14
Londrina, 86051-980 Londrina, PR, Brasil.
Coordenação de Licenciatura em Química (COLIQ), Universidade Tecnológica Federal do Paraná,
Departamento de Bioquímica e Biotecnologia, Centro de Ciências Exatas, Universidade Estadual de
15 16
*Corresponding author: + 55 43 33714080
17
e-mail address: [email protected]
18 19 20
ABSTRACT
21
Biopolymers based hydrogels could have applications in various fields, such as
22
packaging materials, drug delivery systems, biosensors, and agricultural practices. The
23
current work aimed to develop starch/xanthan hydrogels through extrusion and
24
thermopressing processes, using citric acid (CA) as crosslinking agent and sodium
25
hypophosphite (SHP) as catalyst. The hydrogels were produced with different levels of CA
26
(0.00, 0.75, 1.50 and 2.25 g/100 g polymer). Hydration, mechanical, thermal and
27
microstructural properties of the hydrogels were determined. Swelling behavior of the
28
materials with CA was lower than the control. Additionally, CA ensured the preservation of 1
29
hydrogels integrity after the swelling process. Gel fraction increased with CA 0.75 g/100 g.
30
Starch/xanthan hydrogels crosslinked with CA demonstrated lower strength than non-
31
crosslinked hydrogels, which may be related to acid hydrolysis of the polymer chains. CA-
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SHP increased the storage modulus of the hydrogels. Reactive extrusion and
33
thermopressing were efficient methods in the production of crosslinked starch/xanthan
34
based hydrogels.
35 36
Keywords: biopolymer, swelling, tensile strength, sheet, microstructure.
37 38
1. Introduction
39
Environmental concerns over the disposal of non-renewable and non-biodegradable
40
plastics have led to increasing interest in the synthesis and production of biopolymer-
41
based materials (Lambert & Wagner, 2017). These include hydrogels, which are three-
42
dimensionally cross-linked structures, generally highly hydrophilic, that have at same time
43
swelling properties and resistance to dissolution (Ali & Ahmed, 2018). Hydrogels have
44
extended applications in various fields, such as packaging materials (Farris, Schaich, Liu,
45
Piergiovanni & Yam, 2009), controlled drug delivery platforms (Shalviri, Liu, Abdekhodaie
46
& Wu, 2010), biosensors, agricultural systems,etc (Ali & Ahmed, 2018).
47
Starch and xanthan gum are polysaccharides obtained from renewable sources and
48
widely used in the synthesis of biodegradable polymeric materials, including hydrogels
49
(Shalviri, Liu, Abdekhodaie & Wu, 2010; Bueno, Bentini, Catalani,& Petri, 2013; Kim, Choi,
50
Kim, & Lin, 2015; Balasubramanian, Kim,& Lee, 2018).
51
Starch is a homopolysaccharide of glucose, consisting of two structures with great
52
molecular weight: amylose and amylopectin. Amylose consists of essentially linear chains,
53
formed by D-glucose units linked by α-1,4-glycosidic bonds, while amylopectin has a
54
branched structure, with α-1,4 glycosidic bonds in the main chain and α-1,6 at branching 2
55
points (Du, Jia, Xu & Zhou, 2007). When used to produce biodegradable materials, starch
56
is generally combined with a plasticizing agent, such as glycerol, originating thermoplastic
57
starch (TPS) (Chivrac, Pollet & Avérous, 2009).
58
Xanthan gum is an extracellular polysaccharide obtained mainly through the
59
fermentation of Xanthomonas campestris. Its primary structure is constituted by repeated
60
units of glucose, mannose, and glucuronic acid, in the proportion of 2:2:1. Glucuronic acid
61
and pyruvic acid groups on the side chains are responsible for its anionic character (Li et
62
al., 2016).
63
Synthetization of hydrogels from two biopolymers generally improves the stability of
64
the material, impacting on its functionality. The different structures of the polymers may
65
result in a synergism caused by possible conformational changes and chemical
66
interactions between the chains (Liu, Kost, Yan & Spiro, 2012; Balasubramanian, Kim &
67
Lee, 2018; Gong et al., 2019).
68
Polymers chains can be crosslinked via chemical (covalent bonds, ionic
69
interactions, and hydrogen bonds) as well physical interactions (electrostatic, hydrophobic,
70
dipole-dipole, and chains packing) (Tao et al., 2016; Ali & Ahmed, 2018). The resulting
71
hydrogels may have different forms (e.g. films, sheets and coatings).
72
Citric acid (CA) has been widely used as a crosslinking agent in starch (Reddy &
73
Yang, 2010; Olivato, Grossmann, Bilck & Yamashita, 2012; Olivato, Grossmann,
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Yamashita, Eiras & Pessan, 2012, Garcia et al., 2014) and xanthan gum (Bueno, Bentini,
75
Catalani,& Petri, 2013; Tao et al., 2016) materials. However, starch/xanthan gum based
76
hydrogels crosslinked with CA are not reported in the literature.
77
Reddy and Yang (2010) used CA as crosslinking agent associated with sodium
78
hypophosphite (SHP), as catalyst, in starch films produced by casting. These and other
79
authors (Olivato, Grossmann, Bilck & Yamashita, 2012; Garcia et al., 2014) highlighted
80
that, besides the crosslinker function, CA also presents hydrolytic and plasticizing action. 3
81
Although research on hydrogel production by the casting method is quite extensive
82
in the literature, studies reporting the use of processes such as extrusion and
83
thermopressing are limited. Both extrusion and thermopressing subject the materials to
84
thermal and mechanical energy, resulting in chemical and physical reactions (Dastidar &
85
Netravali, 2012; González-Seligra, Ochoa-Yepes, Goyanes & Famá, 2017). Dastidar &
86
Netravali (2012) report the occurrence of a cure process using a hot press and,
87
consequently, an increase in the extent of esterification using malonic acid as a
88
crosslinking agent.
89
The objective of the current study was to investigate the crosslinking effect of CA-
90
SHP on starch/xanthan gum hydrogels produced by extrusion and thermopressing
91
processes.
92 93
2. Material and methods
94
2.1 Materials
95
Cassava starch (18 g/100 g amylose and 82 g/100 g amylopectin) was obtained
96
from Pinduca Food Industry Ltd. (Araruna, Brazil) and xanthan gum Kelzan® was provided
97
by CP Kelco (Limeira, Brazil). Analytical grade CA, SHP, and glycerol were purchased
98
from Synth (Diadema, Brazil).
99 100
2.2 Preparation of starch-xanthan hydrogels
101
Hydrogel sheets were prepared in three stages, according to the formulations
102
shown in Table 1. The levels of CA/SHP were based on Garcia et al. (2014). In the first
103
stage, for the pellets production, CA and SHP were dissolved in glycerol and then
104
manually mixed with the starch/xanthan mixture. The final mixtures were processed using
105
a laboratory single-screw extruder (model EL-25, BGM, São Paulo, Brazil) with a screw
106
diameter (D) of 25 mm and screw length of 26D, through a die with six circular 2 mm 4
107
diameter holes at temperatures of 90/120/130/115 ⁰C, from the feeding zone to the die
108
zone, and a screw speed of 35 rpm. Subsequently, the obtained profiles were pelletized.
109
In the second stage, the pellets were reprocessed in the same equipment, now
110
using a flat die with a 5 mm x 130 mm (thickness and width, respectively) gap, at
111
temperatures of 100/120/130/115 ⁰C and screw speed of 35 rpm, resulting in partially
112
homogeneous sheets.
113
Finally, to obtain the cured hydrogel, approximately 18 g of the extruded pieces
114
were placed between two sheets of cellulose acetate and pressed in a hydraulic press
115
(JOMAQ, model PHB 200, Franca, Brazil) at a temperature of 120 ⁰C. The material was
116
first placed in the equipment without pressure for 6 min and subsequently pressed at 5
117
MPa for 10 min, aiming to prevent the formation of bubbles.
118 119
2.3 Thickness and apparent opacity
120
The thickness was determined using a digital micrometer (1 µm accuracy –
121
Mitutoyo, Kawasaki, Japan). The measurements were repeated at fifteen random positions
122
on each hydrogel formulation. The apparent opacity was determined using a colorimeter
123
(BYK Gardner, Germany) as described by Garcia et al. (2018).
124 125
2.4 Scanning electron microscopy (SEM)
126
The surface and fracture features of the hydrogels were observed using a scanning
127
electron microscope FEI, model Quanta 200 (FEI Company, Tokyo, Japan) as described
128
by Olivato, Grossmann, Bilck & Yamashita (2012).
129 130
2.5 Hydrogels swelling power and gel fraction
131
The swelling power (SP) of the hydrogels was determined according to the method
132
proposed by Yun, Wee, Byun & Yoon (2008) with some modifications. Hydrogel sheets 5
133
were cut into 35 mm diameter samples and conditioned at 53% RH (Mg(NO3)2 saturated
134
solution). Accurately weighed (Wo) samples were immersed in 25 mL of distilled water at
135
room temperature (25 ± 2 ⁰C), for 2 d. Next, the moisture on the surface was removed
136
and the weight of the swelling hydrogels (We) was measured. The swelling power was
137
calculated with the following equation:
138
SP = (We – Wo) / Wo
139
The insoluble portion of the hydrogels was dried in a hot air circulation oven (80
140
⁰C) for 20 h to calculate the gel fraction. The samples were conditioned at 53% RH for 2 d
141
and weighed (Wg). The results were measured in triplicate. The gel fraction (GF) was
142
calculated as follows:
143
GF = Wg / Wo· 100
144 145
2.6 Mechanical properties
146
The tensile properties were determined using the texture analyser model TA.XT
147
plus (Stable Micro Systems, Surrey, UK), according to Garcia et al. (2018) based on the
148
ASTM-D882-02 (2002) standard method.
149 150
2.7 Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy
151
FTIR spectra were collected using a Vertex 70 FTIR spectrophotometer (Bruker,
152
Ettingen, Germany) with a Platinum ATR accessory. An average of 16 scans, recorded
153
from 4000/cm to 400/cm wavenumbers were obtained at a resolution of 4/cm. The
154
samples were conditioned in a desiccator at ≈ 0% RH at 23±2 ⁰C for 10 d before the
155
analysis.
156 157
2.8 Dynamical mechanical analysis (DMA)
6
158
DMA was determined using a dynamic mechanical analyzer, model Q800 (TA
159
Instruments, New Castle, USA). The hydrogel sheets were previously conditioned in a
160
desiccator at 53±2% RH for 7 d. Samples were scanned from -100 to 100 ⁰C with a
161
heating rate of 3 //min and fixed frequency of 1 Hz.
162 163 164 165
2.9 Statistical analysis The data were analyzed using STATISTICA 10.0 software (Statsoft Inc., Tulsa, USA) with analysis of variance (ANOVA) and Tukey's test at a 5% significance level.
166 167
3. Results and discussion
168 169
3.1. Appearance, apparent opacity, and thickness
170
The obtained sheets (hydrogels) presented a smooth surface (Fig. 1). The slightly
171
yellowish color was observed from the initial processing steps and is in accordance with
172
the effect of xanthan and CA in starch-based materials, as described by other authors
173
(Flores, Costa, Yamashita, Gerschenson & Grossmann, 2010; Reddy & Yang, 2010).
174
The apparent opacity of the hydrogels ranged from 0.084 to 0.157% µm-1 (Table 2)
175
and no significant differences were observed between the samples. According to Olivato et
176
al. (2012), generally, crosslinking increases the compaction of the polymer chains,
177
resulting in more opaque films. In the present study this was not observed, although there
178
were indications that crosslinking occurred (as will be discussed later).
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The average thickness (Table 2) of the hydrogels varied from 344 to 525 µm, with
180
significant differences between CA0 and the samples containing CA. The two samples
181
with the highest CA concentration had the lowest thickness. This is due to the fact that CA
182
also has a plasticizing action (Shi et al., 2007) and is capable of performing acid hydrolysis
183
of the polymeric chains, reducing the viscosity of the molten material (Carvalho, Zambon, 7
184
Cuervelo & Gandini, 2005). Thus, the formulations with higher concentrations of CA, when
185
subjected to high pressure during thermopressing, resulted in hydrogels of lower thickness
186
and larger surface.
187 188
3.2. Microstructure
189
Samples CA0, CA1.50, and CA2.25 were homogeneous and smooth on the
190
surface (Fig. 2), a factor that can be attributed to the thermopressing. The partially
191
irregular surface of the sample CA0.75 may be related to a failure of handling event at the
192
time of removing the hydrogel from the acetate sheets after thermopressing.
193
The fracture micrographs presented roughness marks. These were also observed
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by Miranda & Carvalho (2011) in starch films incorporated with CA, who reported them as
195
characteristic of semi-crystalline materials. In our work, the ductility of the materials made
196
the fracture process difficult, it being possible that the fracture was responsible, in part, for
197
the occurrence of this roughness.
198
In the formulation without CA, it is possible to observe circular structures, which
199
seem to be partially intact starch granules. The same structures were not observed in the
200
formulations containing CA, probably because the hydrolytic action of CA favored the
201
destructuration of granules. Olivato, Grossmann, Bilck & Yamashita (2012) also observed
202
partially intact starch granules in starch/poly(butylene adipate-co-terephthalate) (PBAT)
203
films, but these disappeared when CA (1.5 g/100 g) was added.
204 205
3.3. Swelling behavior and gel fraction
206
The water swelling decreased significantly with the addition of CA (Fig.3),
207
however, there were no differences as a function of CA concentration. This reduction may
208
be related to the occurrence of crosslinking, which would make it difficult for water to
209
penetrate into the polymer matrix. The destructuration of the native form of the starch 8
210
granule during the extrusion may have contributed to exposing its hydroxyl groups,
211
allowing greater interaction with the CA molecules. This crosslinking tag, which may also
212
involve xanthan gum, is in accordance with the decreased water swelling profile observed
213
in CA containing hydrogels.
214 215
Dastidar & Netravali (2012) observed a decrease in swelling of potato starch films with the increase in crosslinker concentration.
216
The gel fraction of the hydrogels increased with the addition of 0.75 (g/100 g) of
217
the crosslinking agent (Fig. 3). The decrease in swelling and the increase in the gel
218
fraction for this formulation, when compared to CA0, are indicative of crosslinking.
219
The increase in CA concentration in CA1.50 and CA2.25 hydrogels did not cause
220
further decreases in swelling, compared to the CA0.75 hydrogel. On the other hand, the
221
gel fraction (FG), after increasing in CA0.75, returned to lower values with higher
222
concentrations of CA, probably because there is an ideal (critical) concentration of CA for
223
the formulations and process conditions tested. The addition of CA at concentrations
224
above 0.75 (g/100 g) may cause residual CA, which will act as a plasticizer in the polymer
225
matrix (Shi et al., 2007). This effect, together with the hydrolytic action of CA, would
226
explain the decrease in FG, since the polymer chains would have greater mobility, forming
227
a weaker molecular network.
228
Seligra, Medina Jaramillo, Famá & Goyanes (2016) reported that starch films
229
crosslinked with CA maintained their integrity in the swelling test in DMSO, while the
230
sample without CA was completely solubilized, thus indicating the crosslinking action of
231
CA. A similar phenomenon could be observed in the present work, where hydrogels
232
containing CA remained intact (after immersion in water for 2 d), while the sample without
233
CA was fragmented, partially losing its integrity (Fig.4). The fact that a hydrogel swells
234
without loss of structural integrity arise of great importance for its main applications
235
(membrane, absorbent support, biosensor, molecular carrier, and food packaging). 9
236 237
3.4. Mechanical properties
238
The mechanical properties were affected by the incorporation of CA. The values of
239
the tensile strength (σ) and Young's modulus of the hydrogels with CA were significantly
240
lower than those of CA0 formulation (Table 2). On the other hand, the elongation at break
241
values increased when higher concentrations of CA were used, reaching a 119% increase
242
when comparing CA2.25 with the CA0.
243
The effects of CA on the mechanical properties of starch based materials can be
244
very varied. Thus, Dastidar & Netravali (2012), in their work with potato starch films
245
containing malonic acid (crosslinking agent), reported an increase in Young’s modulus
246
after pre-curing drying and curing thermopressing treatments, which was attributed to the
247
greater crosslinking of the starch after cure. The effect was also attributed to the lower
248
moisture absorption of the materials after crosslinking.
249 250
The contrary effects observed in our work are reported in the literature been result of two main factors: chains degradation under the processing and acid hydrolysis.
251
Extrusion and thermopressing may have a significant effect on the molecular
252
weight of starch materials. Starch polymers suffer significant degradation during extrusion.
253
The molecular weight of the starch polymers significantly affects the physical properties of
254
starch, affecting the rearrangement in the hydrogel network formation (Reddy & Yang,
255
2010; Ali & Ahmed, 2013; Liu, Wang, Yu, Tong, Chen, Liu & Li, 2013).
256
Acid hydrolysis of the polymer chains promoted by CA or its plasticizing action
257
(internal plasticizer) also affected the mechanical properties of starch/xanthan hydrogels.
258
Similar effects to those find in our work have been observed by several authors with CA or
259
other carboxylic acids (Shi et al., 2007; Carvalho, Zambon, Cuervelo & Gandini, 2005; Da
260
Roz et al., 2016).
261 10
262
3.5. Infrared spectroscopy
263
The FTIR-ATR spectra of the hydrogels are shown in Fig.5. All formulations
264
presented similar spectra, except for the bands at 1650/cm and 1723/cm for the samples
265
containing CA, which correspond to the carbonyl stretch (C=O) of carboxylic acid and
266
ester, respectively (Shalviri, Abdekhodaie & Wu, 2010; Dastidar & Netravali, 2012; Bueno,
267
Bentini, Catalani & Petri, 2013; Tao et al., 2016). In the spectrum of the control
268
formulation, it is possible to see the band at 1650/cm although at a lower intensity, due to
269
the presence of a small amount of C=O in the xanthan structure. At the same time, the
270
band near 1650-1640/cm is also assigned to the water adsorbed by the polymer matrix
271
(Dastidar & Netravali, 2012). Considering that the formulations containing CA were
272
washed to remove the unbound reagent, the increase in band intensity at 1650/cm and the
273
presence of the band at 1723/cm indicate the occurrence of a chemical reaction
274
(esterification) between CA and the polymeric chains during reactive extrusion.
275
As a characteristic of the starch and xanthan gum structures, all the formulations
276
showed a broad absorption band at 3400/cm, which is assigned to –OH stretching
277
vibrations (Pavia, 2001). The apparent increase in the intensity of this band may be an
278
indication of the occurrence of hydrolysis of the polymer chains caused by CA.
279
Several studies have used FTIR analysis to confirm the occurrence of crosslinking
280
promoted by polycarboxylic acids. At the same time, other reaction mechanisms have
281
been proposed (Reddy & Yang, 2010; Dastidar & Netravali, 2012; Olivato et al., 2012;
282
Bueno, Bentini, Catalani & Petri, 2013; Garcia et al., 2014; Tao et al., 2016). For the
283
present work, two reaction mechanisms can be considered to represent the occurrence of
284
crosslinking through CA.
285
The first way of explaining how the esterification reaction between starch and
286
xanthan gum may have occurred is based on the mechanisms proposed by Reddy & Yang
287
(2010) and Dastidar & Netravali (2012), with SHP acting as a catalyst (Fig.6). Citric acid, 11
288
when heated, loses a water molecule, giving rise to an anhydride (Scheme 1). With
289
additional heating, another water molecule would be lost. The interaction with the –OH of
290
starch or xanthan gum is favored by these transformations since the carbonyl group of
291
anhydrides is more electrophilic than the acidic group in a nucleophilic addition reaction
292
(Garcia et al., 2014). Thus, the grafting of the citrate group occurs in a starch or xanthan
293
gum chain. In a second step, the grafted functional group may react with the hydroxyl
294
group of another molecule, resulting in a crosslinking. Another route is also possible
295
(Scheme 2), based on the proposal of Bueno, Bentini, Catalani & Petri (2013) for
296
reticulation of xanthan gum by CA. The esterification reaction takes place between groups
297
–COOH present in the structure of xanthan gum and –OH groups of the CA in a first
298
reaction stage, and then, the second stage (crosslinking) follows the same model shown in
299
Scheme 1.
300
The second way that CA may have promoted crosslinking in the presence of SHP
301
is based on the proposals of Yang, Chen, Guan & He (2010), Peng, Yang, Wang e Wang
302
(2012), Garcia et al. (2014), and Garcia et al. (2018) for cellulosic and starchy materials,
303
considering SHP as an agent participating in the reaction. As presented in Scheme 3 (Fig.
304
6), a monoester already formed, as shown in Scheme 1, reacts with SHP and a -HPO2
305
group is grafted in the citrate esterified to the polymer chain. In a second step, an addition
306
reaction between two monoesters results in a crosslinkage. Both starch and xanthan gum
307
molecules could be involved in the esterification reactions. Yang, Chen, Guan & He (2010)
308
demonstrated that this pathway took place at temperatures higher than those described in
309
Schemes 1 and 2 (Fig. 6).
310
Considering the relatively high xanthan gum content, a reaction mechanism
311
without the intervention of CA/SHP could also occur, similar to that proposed by Bueno,
312
Bentini, Catalani & Petri (2013) for the auto esterification between xanthan pyruvyl or
12
313
acetyl groups with OH groups. In the case of the present work, the reaction would also be
314
possible between the -COOH groups of the xanthan and -OH of starch chains.
315 316
3.6. Dynamic mechanical analysis
317
The results of the storage modulus (E’) and loss modulus (tan δ) as a function of
318
the temperature are presented in Fig. 7. For all formulations, decreases in E' values were
319
observed with increasing temperature, with two stages of fall around -60 °C and 20 °C. In
320
the temperature range from -100 to approximately -20 °C, all samples containing CA
321
showed higher E' when compared to the CA0 (Fig. 7a). This behavior indicates lower
322
molecular motility (greater stiffness) in samples containing CA and occurred at
323
temperatures below the Tg of the starch/xanthan-rich phase (see explanation below).
324
The tan δ scans (Fig.7b) presented two relaxation peaks for all formulations, with
325
peaks of higher intensities at temperatures from 11 to 19 ºC, related to the transition of the
326
starch/xanthan-rich phase (associated with Tg) (Averous & Boquillon, 2004; Olivato et al.,
327
2017) and peaks at -60 °C, consistent with the relaxation of the glycerol-rich phase
328
(Averous & Boquillon, 2004).
329
Garcia et al. (2018) observed that extruded films of starch/ PBAT compatibilized
330
with itaconic acid (IA) and SHP presented higher values of storage modulus when
331
compared to control (without IA and SHP), indicating higher stiffness of these samples.
332
Moreover, in the control sample, the authors also observed relaxation peaks related to the
333
phases rich in glycerol and starch, at temperatures of -55 °C and 58 °C, respectively. In
334
the samples with IA-SHP, the Tg of the starch-rich phases were shifted to lower
335
temperatures, indicating depolymerization (hydrolysis) of the starch chains, favoring
336
molecular mobility. Other authors reported similar results using CA in other starch blends
337
(Ortega-Toro, Collazo-Bigliardi, & Talens, 2016) or using malic acid in the same
338
starch/PBAT system (Fahrngruber, Siakkou, Wimmer, Kozich, & Mundigler, 2017). 13
339
In the present work, contrary behavior was observed in the formulations CA0.75
340
and CA1.50, i.e., Tg was shifted to higher temperatures. Thus, despite the hypothesis of
341
acid hydrolysis occurrence, this is indicative of the predominance of chains with less
342
mobility, resulting from crosslinking in the presence of CA/SHP. In the CA2.25 formulation,
343
the profile reported by Garcia et al. (2018) is also observed, with the Tg occurring at a
344
lower temperature when compared to samples with lower concentrations of CA and the
345
control. Finally, in the CA containing samples, a shift in Tg to higher temperatures related
346
to the glycerol-rich phase was observed (Fig. 7b), which is associated with the occurrence
347
of interactions between glycerol and CA/SHP, indicating lower availability of plasticizer in
348
these samples, a phenomenon also reported by Garcia et al. (2018).
349 350
4. Conclusion
351
The production of mixed starch/xanthan gum hydrogels employing CA as
352
crosslinking agent and SHP as catalyst and/or crosslinker, by extrusion followed by
353
thermopressing, is a viable and promising technique. Extrusion allowed good interaction
354
between the components of the hydrogels, promoting crosslinking and characterizing the
355
process as being of reactive extrusion. Thermopressing was responsible for producing
356
homogeneous materials with a smooth surface and good processability. Crosslinking
357
decreases the swelling of the hydrogels while strengthening their structure, making the
358
materials more stable when immersed in water. The CA effect in the mechanical
359
properties, especially the increase in elongation promoted by the hydrolytic action could be
360
beneficial for many applications of hydrogels. Based on the experimental results and the
361
vast literature on the subject, the mechanisms that best represent the crosslinking reaction
362
are those where CA acts as crosslinking agent and SHP can act as a catalyst or as a
363
crosslinker. In order to further investigate the properties and functionalities of the
14
364
hydrogels produced, future works should be carried out on their application in studies of
365
controlled release of different active compounds of interest in the food and drug areas.
366 367
Acknowledgments
368
The authors gratefully acknowledge the financial support received from CAPES
369
(Brazil) through a scholarship to the first author. The authors also express their gratitude to
370
the Multiuser Laboratories Center of PROPPG/UEL for the scanning electron microscopy
371
analysis.
372 373
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Dastidar, T. G., & Netravali, A. N. (2012). “Green” crosslinking of native starches with malonic acid and their properties. Carbohydrate Polymers, 90, 1620–1628. 15
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Flores, S. K., Costa, D., Yamashita, F., Gerschenson, L. N., & Grossmann, M. V. (2010). Mixture design for evaluation of potassium sorbate and xanthan gum effect on properties of tapioca starch films obtained by extrusion. Materials Science and Engineering C, 30, 196–202.
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Seligra, P. G., Medina Jaramillo, C., Famá, L., &Goyanes, S. (2016). Biodegradable and nonretrogradable eco-films based on starch-glycerol with citric acid as crosslinking agent. Carbohydrate Polymers, 138, 66–74.
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Shalviri, A., Liu, Q., Abdekhodaie, M. J., & Wu, X. Y. (2010). Novel modified starch-xanthan gum hydrogels for controlled drug delivery: Synthesis and characterization. Carbohydrate Polymers, 79, 898–907.
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Shi, R., Zhang, Z., Liu, Q., Han, Y., Zhang, L., Chen, D., & Tian, W. (2007). Characterization of citric acid/glycerol co-plasticized thermoplastic starch prepared by melt blending. Carbohydrate Polymers, 69, 748–755.
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Tao, Y., Zhang, R., Xu, W., Bai, Z., Zhou, Y., Zhao, S., Xu, Y., Yu, D. (2016). Rheological behavior and microstructure of release-controlled hydrogels based on xanthan gum crosslinked with sodium trimetaphosphate. Food Hydrocolloids, 52, 923–933.
464 465 466
Yang, C. Q., Chen, D., Guan, J., & He, Q. (2010). Cross-linking cotton cellulose by the combination of maleic acid and sodium hypophosphite. 1. Fabric wrinkle resistance. Industrial and Engineering Chemistry Research, 49, 8325–8332.
467 468 469
Yun, Y. H., Wee, Y. J., Byun, H. S., & Yoon, S. Do. (2008). Biodegradability of chemically modified starch (RS4)/PVA blend films: Part 2. Journal of Polymers and the Environment, 16, 12–18.
470 17
471
Figure captions
472 473
Fig. 1. Appearance of the starch/xanthan hydrogels.
474
Fig. 2. Electronic micrographs of hydrogels surface (above) and fracture (below). Arrows
475
indicate possible partially intact starch granules. Magnification 800x.
476
Fig. 3. Effect of the CA content on the swelling behavior (black round dots) and gel fraction
477
(grey triangles) on the starch/xanthan hydrogels.
478
Fig. 4. Integrity comparison of the hydrogels in the swelling analysis.
479
Fig. 5. FT-IR-ATR spectra of the stach/xanthan hydrogels. CA0 (dotted grey line), CA0.75
480
(dashed grey line), CA1.50 (solid grey line) and CA2.25 (solid black line).
481
Fig. 6. Schemes of reactions between CA/SHP and starch/xanthan forming mono and
482
diesters.
483
Fig. 7. Storage modulus (a) and tan δ (b) of the starch/xanthan hydrogels as function of
484
temperature. CA0 (dotted grey line), CA0.75 (dashed grey line), CA1.50 (solid grey line)
485
and CA2.25 (solid black line).
18
Table 1 Concentration of components in the hydrogel formulations. Formulation Starch
Xanthan
Glycerol
(g)
(g)
(g/100 g of starch + xanthan)
CA0
90
10
30.8
0.0
0.0
CA0.75
90
10
30.8
0.75
0.375
CA1.50
90
10
30.8
1.50
0.750
CA2.25
90
10
30.8
2.25
1.125
CA ( citric acid); SHP (sodium hypophosphite).
CA
SHP
Table 2 Apparent opacity, thickness, tensile strength, strain at break and Young’s modulus of starch/xanthan hydrogels. Sample
Y (% µm-1)
Thickness (µm) a
525 ±38
a
σ (MPa) 3.83 ± 0.33
ε (%) a
63.31 ± 14.82
E (MPa) c
19.25 ± 4.57 a
CA0
0.107 ± 0.019
CA0.75
0.104 ± 0.014 a
430 ± 32 b
2.23 ± 0.24 b
99.49 ± 15.08 b
12.03 ± 2.09 b
CA1.50
0.138 ± 0.058 a
363 ± 25 c
1.49 ± 0.42 c
135.87 ± 28.04 a
8.70 ± 3.47 b
CA2.25
0.148 ± 0.013 a
344 ± 23 c
1.04 ± 0.15 d
138.64 ± 21.94 a
5.22 ± 1.14 c
Y (apparent opacity); σ (tensile strength); ε (strain at break); E (Young’s modulus). a, b, c Different letters in the same column indicate significant differences (p ≤ 0.05) according to Tukey’s test.
Highlights:
Mixed starch/xanthan gum hydrogels were produced using CA/SHP as a crosslinker. Crosslinking preserves the hydrogel integrity even after immersion in water for 48 h. Hydrogels presented lower swelling and higher elongation than the control. Interaction among the components and smooth surface were allowed by processing. Mechanisms were proposed to explain the possible reactions among the components.
Conflicts of interest
The authors state that there is no conflict of interest.
S0023-6438(19)31292-7
DOI:
https://doi.org/10.1016/j.lwt.2019.108950
Reference:
YFSTL 108950
To appear in:
LWT - Food Science and Technology
Received Date: 29 July 2019 Revised Date:
21 November 2019
Accepted Date: 12 December 2019
Please cite this article as: Simões, B.M., Cagnin, C., Yamashita, F., Olivato, J.B., Garcia, Patrí.Salomã., de Oliveira, S.M., Eiras Grossmann, Maria.Victó., Citric acid as crosslinking agent in starch/xanthan gum hydrogels produced by extrusion and thermopressing, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2019.108950. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Bruno Matheus Simões – Hydrogels processing, analysis of hydrogels properties, data analysis and manuscript redaction.
Caroline Cagnin – Hydrogels processing and analysis of some hydrogels properties.
Fábio Yamashita – Manuscript redaction.
Juliana Bonametti Olivato – DMA analysis and data discussion.
Patrícia Salomão Garcia – Manuscript redaction.
Suzana Mali de Oliveira – Manuscript redaction and hydrogels processing.
Maria Victória Eiras Grossmann – Data analysis and manuscript redaction.
1
Citric acid as crosslinking agent in starch/xanthan gum hydrogels produced by
2
extrusion and thermopressing
3 4
Bruno Matheus Simõesa, Caroline Cagnina, Fabio Yamashitaa, Juliana Bonametti Olivatob,
5
Patrícia Salomão Garciac, Suzana Mali de Oliveirad and Maria Victória Eiras Grossmanna*.
6 7
a
8
de Londrina, 86051-980 Londrina, PR, Brasil.
9
b
Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Estadual
Departamento de Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Câmpus Uvaranas,
10
84030-900 Ponta Grossa, PR, Brasil.
11
c
12
Câmpus Apucarana, 86812-460 Apucarana, PR, Brasil.
13
d
14
Londrina, 86051-980 Londrina, PR, Brasil.
Coordenação de Licenciatura em Química (COLIQ), Universidade Tecnológica Federal do Paraná,
Departamento de Bioquímica e Biotecnologia, Centro de Ciências Exatas, Universidade Estadual de
15 16
*Corresponding author: + 55 43 33714080
17
e-mail address: [email protected]
18 19 20
ABSTRACT
21
Biopolymers based hydrogels could have applications in various fields, such as
22
packaging materials, drug delivery systems, biosensors, and agricultural practices. The
23
current work aimed to develop starch/xanthan hydrogels through extrusion and
24
thermopressing processes, using citric acid (CA) as crosslinking agent and sodium
25
hypophosphite (SHP) as catalyst. The hydrogels were produced with different levels of CA
26
(0.00, 0.75, 1.50 and 2.25 g/100 g polymer). Hydration, mechanical, thermal and
27
microstructural properties of the hydrogels were determined. Swelling behavior of the
28
materials with CA was lower than the control. Additionally, CA ensured the preservation of 1
29
hydrogels integrity after the swelling process. Gel fraction increased with CA 0.75 g/100 g.
30
Starch/xanthan hydrogels crosslinked with CA demonstrated lower strength than non-
31
crosslinked hydrogels, which may be related to acid hydrolysis of the polymer chains. CA-
32
SHP increased the storage modulus of the hydrogels. Reactive extrusion and
33
thermopressing were efficient methods in the production of crosslinked starch/xanthan
34
based hydrogels.
35 36
Keywords: biopolymer, swelling, tensile strength, sheet, microstructure.
37 38
1. Introduction
39
Environmental concerns over the disposal of non-renewable and non-biodegradable
40
plastics have led to increasing interest in the synthesis and production of biopolymer-
41
based materials (Lambert & Wagner, 2017). These include hydrogels, which are three-
42
dimensionally cross-linked structures, generally highly hydrophilic, that have at same time
43
swelling properties and resistance to dissolution (Ali & Ahmed, 2018). Hydrogels have
44
extended applications in various fields, such as packaging materials (Farris, Schaich, Liu,
45
Piergiovanni & Yam, 2009), controlled drug delivery platforms (Shalviri, Liu, Abdekhodaie
46
& Wu, 2010), biosensors, agricultural systems,etc (Ali & Ahmed, 2018).
47
Starch and xanthan gum are polysaccharides obtained from renewable sources and
48
widely used in the synthesis of biodegradable polymeric materials, including hydrogels
49
(Shalviri, Liu, Abdekhodaie & Wu, 2010; Bueno, Bentini, Catalani,& Petri, 2013; Kim, Choi,
50
Kim, & Lin, 2015; Balasubramanian, Kim,& Lee, 2018).
51
Starch is a homopolysaccharide of glucose, consisting of two structures with great
52
molecular weight: amylose and amylopectin. Amylose consists of essentially linear chains,
53
formed by D-glucose units linked by α-1,4-glycosidic bonds, while amylopectin has a
54
branched structure, with α-1,4 glycosidic bonds in the main chain and α-1,6 at branching 2
55
points (Du, Jia, Xu & Zhou, 2007). When used to produce biodegradable materials, starch
56
is generally combined with a plasticizing agent, such as glycerol, originating thermoplastic
57
starch (TPS) (Chivrac, Pollet & Avérous, 2009).
58
Xanthan gum is an extracellular polysaccharide obtained mainly through the
59
fermentation of Xanthomonas campestris. Its primary structure is constituted by repeated
60
units of glucose, mannose, and glucuronic acid, in the proportion of 2:2:1. Glucuronic acid
61
and pyruvic acid groups on the side chains are responsible for its anionic character (Li et
62
al., 2016).
63
Synthetization of hydrogels from two biopolymers generally improves the stability of
64
the material, impacting on its functionality. The different structures of the polymers may
65
result in a synergism caused by possible conformational changes and chemical
66
interactions between the chains (Liu, Kost, Yan & Spiro, 2012; Balasubramanian, Kim &
67
Lee, 2018; Gong et al., 2019).
68
Polymers chains can be crosslinked via chemical (covalent bonds, ionic
69
interactions, and hydrogen bonds) as well physical interactions (electrostatic, hydrophobic,
70
dipole-dipole, and chains packing) (Tao et al., 2016; Ali & Ahmed, 2018). The resulting
71
hydrogels may have different forms (e.g. films, sheets and coatings).
72
Citric acid (CA) has been widely used as a crosslinking agent in starch (Reddy &
73
Yang, 2010; Olivato, Grossmann, Bilck & Yamashita, 2012; Olivato, Grossmann,
74
Yamashita, Eiras & Pessan, 2012, Garcia et al., 2014) and xanthan gum (Bueno, Bentini,
75
Catalani,& Petri, 2013; Tao et al., 2016) materials. However, starch/xanthan gum based
76
hydrogels crosslinked with CA are not reported in the literature.
77
Reddy and Yang (2010) used CA as crosslinking agent associated with sodium
78
hypophosphite (SHP), as catalyst, in starch films produced by casting. These and other
79
authors (Olivato, Grossmann, Bilck & Yamashita, 2012; Garcia et al., 2014) highlighted
80
that, besides the crosslinker function, CA also presents hydrolytic and plasticizing action. 3
81
Although research on hydrogel production by the casting method is quite extensive
82
in the literature, studies reporting the use of processes such as extrusion and
83
thermopressing are limited. Both extrusion and thermopressing subject the materials to
84
thermal and mechanical energy, resulting in chemical and physical reactions (Dastidar &
85
Netravali, 2012; González-Seligra, Ochoa-Yepes, Goyanes & Famá, 2017). Dastidar &
86
Netravali (2012) report the occurrence of a cure process using a hot press and,
87
consequently, an increase in the extent of esterification using malonic acid as a
88
crosslinking agent.
89
The objective of the current study was to investigate the crosslinking effect of CA-
90
SHP on starch/xanthan gum hydrogels produced by extrusion and thermopressing
91
processes.
92 93
2. Material and methods
94
2.1 Materials
95
Cassava starch (18 g/100 g amylose and 82 g/100 g amylopectin) was obtained
96
from Pinduca Food Industry Ltd. (Araruna, Brazil) and xanthan gum Kelzan® was provided
97
by CP Kelco (Limeira, Brazil). Analytical grade CA, SHP, and glycerol were purchased
98
from Synth (Diadema, Brazil).
99 100
2.2 Preparation of starch-xanthan hydrogels
101
Hydrogel sheets were prepared in three stages, according to the formulations
102
shown in Table 1. The levels of CA/SHP were based on Garcia et al. (2014). In the first
103
stage, for the pellets production, CA and SHP were dissolved in glycerol and then
104
manually mixed with the starch/xanthan mixture. The final mixtures were processed using
105
a laboratory single-screw extruder (model EL-25, BGM, São Paulo, Brazil) with a screw
106
diameter (D) of 25 mm and screw length of 26D, through a die with six circular 2 mm 4
107
diameter holes at temperatures of 90/120/130/115 ⁰C, from the feeding zone to the die
108
zone, and a screw speed of 35 rpm. Subsequently, the obtained profiles were pelletized.
109
In the second stage, the pellets were reprocessed in the same equipment, now
110
using a flat die with a 5 mm x 130 mm (thickness and width, respectively) gap, at
111
temperatures of 100/120/130/115 ⁰C and screw speed of 35 rpm, resulting in partially
112
homogeneous sheets.
113
Finally, to obtain the cured hydrogel, approximately 18 g of the extruded pieces
114
were placed between two sheets of cellulose acetate and pressed in a hydraulic press
115
(JOMAQ, model PHB 200, Franca, Brazil) at a temperature of 120 ⁰C. The material was
116
first placed in the equipment without pressure for 6 min and subsequently pressed at 5
117
MPa for 10 min, aiming to prevent the formation of bubbles.
118 119
2.3 Thickness and apparent opacity
120
The thickness was determined using a digital micrometer (1 µm accuracy –
121
Mitutoyo, Kawasaki, Japan). The measurements were repeated at fifteen random positions
122
on each hydrogel formulation. The apparent opacity was determined using a colorimeter
123
(BYK Gardner, Germany) as described by Garcia et al. (2018).
124 125
2.4 Scanning electron microscopy (SEM)
126
The surface and fracture features of the hydrogels were observed using a scanning
127
electron microscope FEI, model Quanta 200 (FEI Company, Tokyo, Japan) as described
128
by Olivato, Grossmann, Bilck & Yamashita (2012).
129 130
2.5 Hydrogels swelling power and gel fraction
131
The swelling power (SP) of the hydrogels was determined according to the method
132
proposed by Yun, Wee, Byun & Yoon (2008) with some modifications. Hydrogel sheets 5
133
were cut into 35 mm diameter samples and conditioned at 53% RH (Mg(NO3)2 saturated
134
solution). Accurately weighed (Wo) samples were immersed in 25 mL of distilled water at
135
room temperature (25 ± 2 ⁰C), for 2 d. Next, the moisture on the surface was removed
136
and the weight of the swelling hydrogels (We) was measured. The swelling power was
137
calculated with the following equation:
138
SP = (We – Wo) / Wo
139
The insoluble portion of the hydrogels was dried in a hot air circulation oven (80
140
⁰C) for 20 h to calculate the gel fraction. The samples were conditioned at 53% RH for 2 d
141
and weighed (Wg). The results were measured in triplicate. The gel fraction (GF) was
142
calculated as follows:
143
GF = Wg / Wo· 100
144 145
2.6 Mechanical properties
146
The tensile properties were determined using the texture analyser model TA.XT
147
plus (Stable Micro Systems, Surrey, UK), according to Garcia et al. (2018) based on the
148
ASTM-D882-02 (2002) standard method.
149 150
2.7 Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy
151
FTIR spectra were collected using a Vertex 70 FTIR spectrophotometer (Bruker,
152
Ettingen, Germany) with a Platinum ATR accessory. An average of 16 scans, recorded
153
from 4000/cm to 400/cm wavenumbers were obtained at a resolution of 4/cm. The
154
samples were conditioned in a desiccator at ≈ 0% RH at 23±2 ⁰C for 10 d before the
155
analysis.
156 157
2.8 Dynamical mechanical analysis (DMA)
6
158
DMA was determined using a dynamic mechanical analyzer, model Q800 (TA
159
Instruments, New Castle, USA). The hydrogel sheets were previously conditioned in a
160
desiccator at 53±2% RH for 7 d. Samples were scanned from -100 to 100 ⁰C with a
161
heating rate of 3 //min and fixed frequency of 1 Hz.
162 163 164 165
2.9 Statistical analysis The data were analyzed using STATISTICA 10.0 software (Statsoft Inc., Tulsa, USA) with analysis of variance (ANOVA) and Tukey's test at a 5% significance level.
166 167
3. Results and discussion
168 169
3.1. Appearance, apparent opacity, and thickness
170
The obtained sheets (hydrogels) presented a smooth surface (Fig. 1). The slightly
171
yellowish color was observed from the initial processing steps and is in accordance with
172
the effect of xanthan and CA in starch-based materials, as described by other authors
173
(Flores, Costa, Yamashita, Gerschenson & Grossmann, 2010; Reddy & Yang, 2010).
174
The apparent opacity of the hydrogels ranged from 0.084 to 0.157% µm-1 (Table 2)
175
and no significant differences were observed between the samples. According to Olivato et
176
al. (2012), generally, crosslinking increases the compaction of the polymer chains,
177
resulting in more opaque films. In the present study this was not observed, although there
178
were indications that crosslinking occurred (as will be discussed later).
179
The average thickness (Table 2) of the hydrogels varied from 344 to 525 µm, with
180
significant differences between CA0 and the samples containing CA. The two samples
181
with the highest CA concentration had the lowest thickness. This is due to the fact that CA
182
also has a plasticizing action (Shi et al., 2007) and is capable of performing acid hydrolysis
183
of the polymeric chains, reducing the viscosity of the molten material (Carvalho, Zambon, 7
184
Cuervelo & Gandini, 2005). Thus, the formulations with higher concentrations of CA, when
185
subjected to high pressure during thermopressing, resulted in hydrogels of lower thickness
186
and larger surface.
187 188
3.2. Microstructure
189
Samples CA0, CA1.50, and CA2.25 were homogeneous and smooth on the
190
surface (Fig. 2), a factor that can be attributed to the thermopressing. The partially
191
irregular surface of the sample CA0.75 may be related to a failure of handling event at the
192
time of removing the hydrogel from the acetate sheets after thermopressing.
193
The fracture micrographs presented roughness marks. These were also observed
194
by Miranda & Carvalho (2011) in starch films incorporated with CA, who reported them as
195
characteristic of semi-crystalline materials. In our work, the ductility of the materials made
196
the fracture process difficult, it being possible that the fracture was responsible, in part, for
197
the occurrence of this roughness.
198
In the formulation without CA, it is possible to observe circular structures, which
199
seem to be partially intact starch granules. The same structures were not observed in the
200
formulations containing CA, probably because the hydrolytic action of CA favored the
201
destructuration of granules. Olivato, Grossmann, Bilck & Yamashita (2012) also observed
202
partially intact starch granules in starch/poly(butylene adipate-co-terephthalate) (PBAT)
203
films, but these disappeared when CA (1.5 g/100 g) was added.
204 205
3.3. Swelling behavior and gel fraction
206
The water swelling decreased significantly with the addition of CA (Fig.3),
207
however, there were no differences as a function of CA concentration. This reduction may
208
be related to the occurrence of crosslinking, which would make it difficult for water to
209
penetrate into the polymer matrix. The destructuration of the native form of the starch 8
210
granule during the extrusion may have contributed to exposing its hydroxyl groups,
211
allowing greater interaction with the CA molecules. This crosslinking tag, which may also
212
involve xanthan gum, is in accordance with the decreased water swelling profile observed
213
in CA containing hydrogels.
214 215
Dastidar & Netravali (2012) observed a decrease in swelling of potato starch films with the increase in crosslinker concentration.
216
The gel fraction of the hydrogels increased with the addition of 0.75 (g/100 g) of
217
the crosslinking agent (Fig. 3). The decrease in swelling and the increase in the gel
218
fraction for this formulation, when compared to CA0, are indicative of crosslinking.
219
The increase in CA concentration in CA1.50 and CA2.25 hydrogels did not cause
220
further decreases in swelling, compared to the CA0.75 hydrogel. On the other hand, the
221
gel fraction (FG), after increasing in CA0.75, returned to lower values with higher
222
concentrations of CA, probably because there is an ideal (critical) concentration of CA for
223
the formulations and process conditions tested. The addition of CA at concentrations
224
above 0.75 (g/100 g) may cause residual CA, which will act as a plasticizer in the polymer
225
matrix (Shi et al., 2007). This effect, together with the hydrolytic action of CA, would
226
explain the decrease in FG, since the polymer chains would have greater mobility, forming
227
a weaker molecular network.
228
Seligra, Medina Jaramillo, Famá & Goyanes (2016) reported that starch films
229
crosslinked with CA maintained their integrity in the swelling test in DMSO, while the
230
sample without CA was completely solubilized, thus indicating the crosslinking action of
231
CA. A similar phenomenon could be observed in the present work, where hydrogels
232
containing CA remained intact (after immersion in water for 2 d), while the sample without
233
CA was fragmented, partially losing its integrity (Fig.4). The fact that a hydrogel swells
234
without loss of structural integrity arise of great importance for its main applications
235
(membrane, absorbent support, biosensor, molecular carrier, and food packaging). 9
236 237
3.4. Mechanical properties
238
The mechanical properties were affected by the incorporation of CA. The values of
239
the tensile strength (σ) and Young's modulus of the hydrogels with CA were significantly
240
lower than those of CA0 formulation (Table 2). On the other hand, the elongation at break
241
values increased when higher concentrations of CA were used, reaching a 119% increase
242
when comparing CA2.25 with the CA0.
243
The effects of CA on the mechanical properties of starch based materials can be
244
very varied. Thus, Dastidar & Netravali (2012), in their work with potato starch films
245
containing malonic acid (crosslinking agent), reported an increase in Young’s modulus
246
after pre-curing drying and curing thermopressing treatments, which was attributed to the
247
greater crosslinking of the starch after cure. The effect was also attributed to the lower
248
moisture absorption of the materials after crosslinking.
249 250
The contrary effects observed in our work are reported in the literature been result of two main factors: chains degradation under the processing and acid hydrolysis.
251
Extrusion and thermopressing may have a significant effect on the molecular
252
weight of starch materials. Starch polymers suffer significant degradation during extrusion.
253
The molecular weight of the starch polymers significantly affects the physical properties of
254
starch, affecting the rearrangement in the hydrogel network formation (Reddy & Yang,
255
2010; Ali & Ahmed, 2013; Liu, Wang, Yu, Tong, Chen, Liu & Li, 2013).
256
Acid hydrolysis of the polymer chains promoted by CA or its plasticizing action
257
(internal plasticizer) also affected the mechanical properties of starch/xanthan hydrogels.
258
Similar effects to those find in our work have been observed by several authors with CA or
259
other carboxylic acids (Shi et al., 2007; Carvalho, Zambon, Cuervelo & Gandini, 2005; Da
260
Roz et al., 2016).
261 10
262
3.5. Infrared spectroscopy
263
The FTIR-ATR spectra of the hydrogels are shown in Fig.5. All formulations
264
presented similar spectra, except for the bands at 1650/cm and 1723/cm for the samples
265
containing CA, which correspond to the carbonyl stretch (C=O) of carboxylic acid and
266
ester, respectively (Shalviri, Abdekhodaie & Wu, 2010; Dastidar & Netravali, 2012; Bueno,
267
Bentini, Catalani & Petri, 2013; Tao et al., 2016). In the spectrum of the control
268
formulation, it is possible to see the band at 1650/cm although at a lower intensity, due to
269
the presence of a small amount of C=O in the xanthan structure. At the same time, the
270
band near 1650-1640/cm is also assigned to the water adsorbed by the polymer matrix
271
(Dastidar & Netravali, 2012). Considering that the formulations containing CA were
272
washed to remove the unbound reagent, the increase in band intensity at 1650/cm and the
273
presence of the band at 1723/cm indicate the occurrence of a chemical reaction
274
(esterification) between CA and the polymeric chains during reactive extrusion.
275
As a characteristic of the starch and xanthan gum structures, all the formulations
276
showed a broad absorption band at 3400/cm, which is assigned to –OH stretching
277
vibrations (Pavia, 2001). The apparent increase in the intensity of this band may be an
278
indication of the occurrence of hydrolysis of the polymer chains caused by CA.
279
Several studies have used FTIR analysis to confirm the occurrence of crosslinking
280
promoted by polycarboxylic acids. At the same time, other reaction mechanisms have
281
been proposed (Reddy & Yang, 2010; Dastidar & Netravali, 2012; Olivato et al., 2012;
282
Bueno, Bentini, Catalani & Petri, 2013; Garcia et al., 2014; Tao et al., 2016). For the
283
present work, two reaction mechanisms can be considered to represent the occurrence of
284
crosslinking through CA.
285
The first way of explaining how the esterification reaction between starch and
286
xanthan gum may have occurred is based on the mechanisms proposed by Reddy & Yang
287
(2010) and Dastidar & Netravali (2012), with SHP acting as a catalyst (Fig.6). Citric acid, 11
288
when heated, loses a water molecule, giving rise to an anhydride (Scheme 1). With
289
additional heating, another water molecule would be lost. The interaction with the –OH of
290
starch or xanthan gum is favored by these transformations since the carbonyl group of
291
anhydrides is more electrophilic than the acidic group in a nucleophilic addition reaction
292
(Garcia et al., 2014). Thus, the grafting of the citrate group occurs in a starch or xanthan
293
gum chain. In a second step, the grafted functional group may react with the hydroxyl
294
group of another molecule, resulting in a crosslinking. Another route is also possible
295
(Scheme 2), based on the proposal of Bueno, Bentini, Catalani & Petri (2013) for
296
reticulation of xanthan gum by CA. The esterification reaction takes place between groups
297
–COOH present in the structure of xanthan gum and –OH groups of the CA in a first
298
reaction stage, and then, the second stage (crosslinking) follows the same model shown in
299
Scheme 1.
300
The second way that CA may have promoted crosslinking in the presence of SHP
301
is based on the proposals of Yang, Chen, Guan & He (2010), Peng, Yang, Wang e Wang
302
(2012), Garcia et al. (2014), and Garcia et al. (2018) for cellulosic and starchy materials,
303
considering SHP as an agent participating in the reaction. As presented in Scheme 3 (Fig.
304
6), a monoester already formed, as shown in Scheme 1, reacts with SHP and a -HPO2
305
group is grafted in the citrate esterified to the polymer chain. In a second step, an addition
306
reaction between two monoesters results in a crosslinkage. Both starch and xanthan gum
307
molecules could be involved in the esterification reactions. Yang, Chen, Guan & He (2010)
308
demonstrated that this pathway took place at temperatures higher than those described in
309
Schemes 1 and 2 (Fig. 6).
310
Considering the relatively high xanthan gum content, a reaction mechanism
311
without the intervention of CA/SHP could also occur, similar to that proposed by Bueno,
312
Bentini, Catalani & Petri (2013) for the auto esterification between xanthan pyruvyl or
12
313
acetyl groups with OH groups. In the case of the present work, the reaction would also be
314
possible between the -COOH groups of the xanthan and -OH of starch chains.
315 316
3.6. Dynamic mechanical analysis
317
The results of the storage modulus (E’) and loss modulus (tan δ) as a function of
318
the temperature are presented in Fig. 7. For all formulations, decreases in E' values were
319
observed with increasing temperature, with two stages of fall around -60 °C and 20 °C. In
320
the temperature range from -100 to approximately -20 °C, all samples containing CA
321
showed higher E' when compared to the CA0 (Fig. 7a). This behavior indicates lower
322
molecular motility (greater stiffness) in samples containing CA and occurred at
323
temperatures below the Tg of the starch/xanthan-rich phase (see explanation below).
324
The tan δ scans (Fig.7b) presented two relaxation peaks for all formulations, with
325
peaks of higher intensities at temperatures from 11 to 19 ºC, related to the transition of the
326
starch/xanthan-rich phase (associated with Tg) (Averous & Boquillon, 2004; Olivato et al.,
327
2017) and peaks at -60 °C, consistent with the relaxation of the glycerol-rich phase
328
(Averous & Boquillon, 2004).
329
Garcia et al. (2018) observed that extruded films of starch/ PBAT compatibilized
330
with itaconic acid (IA) and SHP presented higher values of storage modulus when
331
compared to control (without IA and SHP), indicating higher stiffness of these samples.
332
Moreover, in the control sample, the authors also observed relaxation peaks related to the
333
phases rich in glycerol and starch, at temperatures of -55 °C and 58 °C, respectively. In
334
the samples with IA-SHP, the Tg of the starch-rich phases were shifted to lower
335
temperatures, indicating depolymerization (hydrolysis) of the starch chains, favoring
336
molecular mobility. Other authors reported similar results using CA in other starch blends
337
(Ortega-Toro, Collazo-Bigliardi, & Talens, 2016) or using malic acid in the same
338
starch/PBAT system (Fahrngruber, Siakkou, Wimmer, Kozich, & Mundigler, 2017). 13
339
In the present work, contrary behavior was observed in the formulations CA0.75
340
and CA1.50, i.e., Tg was shifted to higher temperatures. Thus, despite the hypothesis of
341
acid hydrolysis occurrence, this is indicative of the predominance of chains with less
342
mobility, resulting from crosslinking in the presence of CA/SHP. In the CA2.25 formulation,
343
the profile reported by Garcia et al. (2018) is also observed, with the Tg occurring at a
344
lower temperature when compared to samples with lower concentrations of CA and the
345
control. Finally, in the CA containing samples, a shift in Tg to higher temperatures related
346
to the glycerol-rich phase was observed (Fig. 7b), which is associated with the occurrence
347
of interactions between glycerol and CA/SHP, indicating lower availability of plasticizer in
348
these samples, a phenomenon also reported by Garcia et al. (2018).
349 350
4. Conclusion
351
The production of mixed starch/xanthan gum hydrogels employing CA as
352
crosslinking agent and SHP as catalyst and/or crosslinker, by extrusion followed by
353
thermopressing, is a viable and promising technique. Extrusion allowed good interaction
354
between the components of the hydrogels, promoting crosslinking and characterizing the
355
process as being of reactive extrusion. Thermopressing was responsible for producing
356
homogeneous materials with a smooth surface and good processability. Crosslinking
357
decreases the swelling of the hydrogels while strengthening their structure, making the
358
materials more stable when immersed in water. The CA effect in the mechanical
359
properties, especially the increase in elongation promoted by the hydrolytic action could be
360
beneficial for many applications of hydrogels. Based on the experimental results and the
361
vast literature on the subject, the mechanisms that best represent the crosslinking reaction
362
are those where CA acts as crosslinking agent and SHP can act as a catalyst or as a
363
crosslinker. In order to further investigate the properties and functionalities of the
14
364
hydrogels produced, future works should be carried out on their application in studies of
365
controlled release of different active compounds of interest in the food and drug areas.
366 367
Acknowledgments
368
The authors gratefully acknowledge the financial support received from CAPES
369
(Brazil) through a scholarship to the first author. The authors also express their gratitude to
370
the Multiuser Laboratories Center of PROPPG/UEL for the scanning electron microscopy
371
analysis.
372 373
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470 17
471
Figure captions
472 473
Fig. 1. Appearance of the starch/xanthan hydrogels.
474
Fig. 2. Electronic micrographs of hydrogels surface (above) and fracture (below). Arrows
475
indicate possible partially intact starch granules. Magnification 800x.
476
Fig. 3. Effect of the CA content on the swelling behavior (black round dots) and gel fraction
477
(grey triangles) on the starch/xanthan hydrogels.
478
Fig. 4. Integrity comparison of the hydrogels in the swelling analysis.
479
Fig. 5. FT-IR-ATR spectra of the stach/xanthan hydrogels. CA0 (dotted grey line), CA0.75
480
(dashed grey line), CA1.50 (solid grey line) and CA2.25 (solid black line).
481
Fig. 6. Schemes of reactions between CA/SHP and starch/xanthan forming mono and
482
diesters.
483
Fig. 7. Storage modulus (a) and tan δ (b) of the starch/xanthan hydrogels as function of
484
temperature. CA0 (dotted grey line), CA0.75 (dashed grey line), CA1.50 (solid grey line)
485
and CA2.25 (solid black line).
18
Table 1 Concentration of components in the hydrogel formulations. Formulation Starch
Xanthan
Glycerol
(g)
(g)
(g/100 g of starch + xanthan)
CA0
90
10
30.8
0.0
0.0
CA0.75
90
10
30.8
0.75
0.375
CA1.50
90
10
30.8
1.50
0.750
CA2.25
90
10
30.8
2.25
1.125
CA ( citric acid); SHP (sodium hypophosphite).
CA
SHP
Table 2 Apparent opacity, thickness, tensile strength, strain at break and Young’s modulus of starch/xanthan hydrogels. Sample
Y (% µm-1)
Thickness (µm) a
525 ±38
a
σ (MPa) 3.83 ± 0.33
ε (%) a
63.31 ± 14.82
E (MPa) c
19.25 ± 4.57 a
CA0
0.107 ± 0.019
CA0.75
0.104 ± 0.014 a
430 ± 32 b
2.23 ± 0.24 b
99.49 ± 15.08 b
12.03 ± 2.09 b
CA1.50
0.138 ± 0.058 a
363 ± 25 c
1.49 ± 0.42 c
135.87 ± 28.04 a
8.70 ± 3.47 b
CA2.25
0.148 ± 0.013 a
344 ± 23 c
1.04 ± 0.15 d
138.64 ± 21.94 a
5.22 ± 1.14 c
Y (apparent opacity); σ (tensile strength); ε (strain at break); E (Young’s modulus). a, b, c Different letters in the same column indicate significant differences (p ≤ 0.05) according to Tukey’s test.
Highlights:
Mixed starch/xanthan gum hydrogels were produced using CA/SHP as a crosslinker. Crosslinking preserves the hydrogel integrity even after immersion in water for 48 h. Hydrogels presented lower swelling and higher elongation than the control. Interaction among the components and smooth surface were allowed by processing. Mechanisms were proposed to explain the possible reactions among the components.
Conflicts of interest
The authors state that there is no conflict of interest.