Journal of Thermal Analysis and Calorimetry

, Volume 133, Issue 1, pp 549–557 | Cite as

Influence of the addition of hydrocolloids on the thermal, pasting and structural properties of starch from common vetch seeds (Vicia sativa sp)

  • Camila Delinski Bet
  • Cristina Soltovski de Oliveira
  • Cleoci Beninca
  • Tiago André Denck Colman
  • Luiz Gustavo Lacerda
  • Egon Schnitzler


Belonging to the leguminous family, the common vetch seeds are a non-conventional starch source that can be exploited to attend the growing demand of the food industry. Thus, the aim of this study was to evaluate the influence of addition of hydrocolloids (5% of: carrageenan, carboxymethylcellulose (CMC), guar, jatahy, xanthan and pectin) on the thermal, pasting and structural properties of starch from common vetch (Vicia sativa) seeds. The interaction with the hydrocolloids was analysed by thermogravimetry and derivative thermogravimetry, differential scanning calorimetry (DSC), viscoamylographic analysis (RVA) and X-ray powder diffraction (DRX). From the thermogravimetry, a similar behaviour was observed for all the studied samples, with three well-defined mass losses and a thermal stability plateau. The addition of guar gum promoted an increase in its thermal stability, unlike the sample added of CMC. From the DSC analysis, it was possible to observe a decrease in onset temperatures with the addition of hydrocolloids, which can be correlated with the decrease in pasting temperatures obtained by RVA, except for the carrageenan gum, which increased these temperatures. By RVA an increase in peak viscosities was achieved, highlighting the addition of CMC, pectin and xanthan. Small variations in the degree of relative crystallinity were noted, but no changes in the diffraction pattern occurred for the samples from the XRD analysis. Although native starch has some limitations, the synergistic effect produced by the addition of gums can optimise its technological properties expanding the industrial use.


Common vetch starch Hydrocolloids Thermal analysis Pasting properties 


To improve the quality of the final products, the additives are important allies of the food industry. Among the options, the hydrocolloids stand out because they act in the improvement of the technological or functional properties of the foods, being classified as polysaccharides of high molecular weight and soluble in water [1, 2]. The gums, belonging to the class of hydrocolloids, when in solution, have high polymeric nature and good interaction between the chains, improving the mouth feel and solution viscosity. These present thickening and gelling functions [3, 4].

In starch and starchy foods, the addition of gums can alter the stability and rheological properties, minimising retrogradation and optimising water retention capacity and solubility, as well as stability to freezing and thawing. This extends the use of starch in the food and pharmaceutical industries. The effects resulting from the addition of gums in the starch depend directly on the hydrocolloid used, the mixing ratio and the molecular weight of the constituents. The breads industry has added gums in the formulation to promote improvements in the volume and texture of these products and as a gluten substitute [5, 6].

The hydrocolloids interact with the starch during gelatinization, where a continuous phase containing leached amylose and a dispersed phase containing fragmented granules and amylopectin can be identified. The presence of hydrocolloids alters the continuous phase [7], interacting with amylose solubilised and with short chains of amylopectin [8]. When the starch gelatinises, the granules swell by reducing the space of the gums present in the continuous phase, increasing their concentration and consequently the viscosity of the suspension. Thus, the addition of gums may alter the gelatinisation process of the starch due to the morphology of the gum; swelling power between the granules and electrostatic interaction between starch and gums as explained by Chaisawang and Suphantharika [9].

Polysaccharides such as pectin and guar, carrageenan, arabic and xanthan gums are the major hydrocolloids applied in the food industry and studied in combination with different varieties of starches [10], but there are still no studies reporting the influence of these hydrocolloids on common vetch starch.

Starch is a storage polysaccharide which consists of amylose and amylopectin chains formed from glucose residues. Amylose is a linear chain in which the glucose molecules are bonded by links α 1 → 4 with few branches. The branched region of the starch refers to amylopectin, which has a main linear chain with several branches through glycosidic linkages α 1 → 6 [11]. The most common sources for obtaining starch are corn, wheat, cassava and potato. However, in order to achieve specific technological properties, the study of new starch sources is extremely important [12, 13].

The seeds of common vetch (Vicia sativa sp) are an unexplored source for the extraction of starch, because its main use is as a source of protein (30% on a dry basis in the seed). However, it is possible to expand its industrial application since the plant adapts easily to the different planting conditions, with its main producers: Asia, Africa and Europe [14]. It is estimated 50% content of starch in vetch seeds [15] and in our previous studies [16], the native common vetch starch presented an oval shape, with an average size of 23.98 µm, gelatinisation temperature rate between 65.07–71.68 °C and lower viscosity (796.05 mPa s), making interesting the study of the properties that can be reached after the addition of gums.

Several studies have already reported a synergistic effect of hydrocolloids on the properties of starch, as well as on: wheat and potato starch blends [17]; cowpea starch [18], pea starch [19]; rice starch [20], normal, waxy and high-amylose corn starches [21]; cationic tapioca starch [22].

However, it is important to investigate which changes are produced by different gums in common vetch starch, since this study is pioneer in the application of gums in this starch source. Thus, the aim of this study was to evaluate the influence of addition of different hydrocolloids on the thermal, structural, and pasting properties of starch from common vetch (Vicia sativa sp).

Materials and methods

Raw materials

The common vetch seeds were acquired in a local market (Imbituva, Paraná, Brazil). All hydrocolloids used in this study were food grade. The following hydrocolloids were used: carrageenan (Starmix, KERRY®), carboxymethylcellulose (Starmix CS, KERRY™), guar (Starmix YG 142, KERRY™), jatahy (Starmix YG 314, KERRY™), pectin (Grindsted™ Pectin LA 110, DANISCO™) and xanthan (Starmix MS 480, KERRY™).

Starch extraction

Flour was produced from the common vetch seeds crushed in a mill (Ika Werke M20, Wilmington, North Carolina, USA). The flour was suspended in water (1:4 w/w, water:starch) and kept under stirring for 10 min and then passed through two sieves of 150 and 270 mesh, consecutively. The filtrate was allowed to stand under refrigeration for decantation of the starch. After 1 h, the supernatant was discarded and the starch centrifuged (Rotina 420R, Germany) at 9000 rpm for 5 min. A scraping was performed to remove a darker layer deposited on the starch. The starch was recovered and dried in a circulating air oven (TE 394/1 Tecnal, São Paulo, BR) at 40° C for 24 h.

Starch modification

A physical mixture of the powdered components containing five per cent (5%, dry basis) of hydrocolloids, and common vetch starch was made and homogenised for 5 min by means of pistil and mortar. The samples were kept in desiccator with anhydrous calcium chloride until analysis. The sample (a) corresponds to the native starch, in which there was no addition of gum, and the other samples were added of the following hydrocolloids: (b) carrageenan; (c) carboxymethylcellulose (CMC); (d) guar; (e) jatahy; (f) pectin; and (g) xanthan.

Thermal analysis

Thermogravimetry (TG)

Thermal analysis system TGA-50 (Shimadzu, Japan), previously calibrated using standard calcium oxalate monohydrate, was used to obtain thermogravimetric and derivative thermogravimetric curves (TG/DTG). TG curves were obtained from 10.0 to 14.0 mg of each sample weighed in open alumina crucibles and subjected to a heating from 30 to 650 °C, under air flow of 150 mL min−1 at a heating rate of 10 °C min−1. The derivative thermogravimetric curves (DTG) and all the percentages of mass loss were established using TA-60 WS data analysis software [16].

Differential scanning calorimetry (DSC)

Starch dispersions were prepared with approximately 2.5 mg of starch and 10 μL of distilled water in sealed aluminium crucibles. The conditions of the analysis were: temperature range from 30 to 100 °C, air flow of 50 mL min−1 at a heating rate of 10 °C min−1. The instrument was previously calibrated with 99.99% purity standard indium, m.p. = 156.6 °C, ΔH = 28.56 J g−1. The DSC curves were obtained using a DSC-Q200 (TA-Instruments) thermal analysis system [23].

Viscosity analysis (RVA)

A mass of each mixture starch/gum (2.24 g, dry basis) was weighed, and the volume was completed to 28 g with distilled water. A viscosimeter Rapid Visco Analyzer RVA-4 (Newport Scientific, Australia) was used to determine the pasting properties. This suspension was subjected to a heating and cooling cycle, where the sample was held at 50 °C for 2 min, heated from 50 to 95 °C at 6 °C min−1, and then held at 95 °C for 5 min. Finally, the sample was cooled to 50 °C at 6 °C min−1 and held at 50 °C for 2 min [24].

X-ray powder diffraction (DRX)

X-ray diffractometer (Ultima 4, Rigaku, Japan) employing CuKα radiation (λ = 1.5418 Å) and settings of 40 kV and 30 mA was used to obtain X-ray diffraction patterns of the samples. The scattered radiation was detected in the angular range of 5°–50° (2θ), with a scanning speed of 2° min−1 and a step of 0.02°. The degree of relative crystallinity was quantitatively estimated following the method described in the literature [25].

Statistical analysis

Assuming the normality of the samples, the homoscedasticity of the variances was tested by Levene’s test (p > 0.05) and applied ANOVA followed by Tukey’s test to identified differences between the samples adopting 95% confidence (p < 0.05). Action software (version was used for data analysis.

Results and discussion

Thermal analysis

Thermogravimetry and derivative thermogravimetry (TG/DTG)

The TG/DTG curves are shown in Fig. 1. All the samples showed three well-defined mass losses when subjected to a temperature increase. The dehydration corresponds to the first mass loss according by Pumacahua-Ramos et al. [26]. The anhydrous samples showed a stability plateau, which is an important parameter for thermal processes.
Fig. 1

TG/DTG curves for native common vetch starch (a) and its mixture with: b carrageenan, c carboxymethylcellulose (CMC), d guar, e jatahy, f pectin and g xanthan

The second mass loss was related to the decomposition of the starch due to the depolymerisation of the amylose and amylopectin chains formed by glucose units at temperatures above 300 °C [27]. In addition, the hydrocolloids degradation also occurs, since they are also polysaccharides formed by different sugar molecules, which undergo decomposition in temperatures around 200 °C [28]. The third mass loss was associated to sample oxidation, since this analysis occurred under air flow.

From the data extracted from the TG curves, which are given in Table 1, it is possible to observe that the water mass contained in the native sample was 12.17%, and between 8.70 and 10.61% for the starch-hydrocolloid systems. This step is dependent on the drying process after the starch is extracted, the heat exchange area, as well as the amount of water bound, since the drying temperature does not exceed 40° C, in addition to the time and storage conditions of the samples until the analysis. The temperature range in which the dehydration of the samples occurred was 30–153 °C.
Table 1

Results obtained from TG/DTG curves for native common vetch starch (a) and its mixture with: (b) carrageenan, (c) carboxymethylcellulose (CMC), (d) guar, (e) jatahy, (f) pectin and (g) xanthan


TG results

DTG results














































































































m, mass loss (%); ∆T, temperature range (°C); Tp, peak temperature (°C)

An increase in the thermal stability of all the samples compared to the native one was verified. Guar gum, a neutral polysaccharide, caused the greatest increase in the thermal stability temperature of common vetch starch, followed by pectin and xanthan. Alberton et al. [29] reported higher thermal stability for cassava starch after addition of jatahy, unlike this study in which this gum slightly increased this stability plateau.

The second step of mass loss corresponds to the depolymerisation of starch/hydrocolloids systems, which presented maximum loss rate at peak temperatures between 288 and 305 °C. The decomposition temperatures found for the common vetch starch associated with CMC are in accordance with those reported by Han et al. [30] for this gum. In other studies by thermal analysis [31], guar gum presented initial decomposition temperature of 246 °C, with cleavage of glycosidic bonds of the mannose and galactose chains; and the maximum weight loss occurred at 300 °C, such as obtained in this study for the initial temperature of second mass loss and the peak temperature calculated by DTG curve (sample d). The xanthan gum showed initial decomposition temperature at 230 °C [32], similar temperature obtained for the beginning of the second mass loss of the common vetch starch combined with this gum (sample g).

The complete oxidation of the starch/hydrocolloid system occurred at temperatures slightly higher than the native sample, until the formation of ash, which corresponded to the following values: (a) 1.15%; (b) 1.88%; (c) 0.55%; (d) 0.42%; (e) 0.83%; (f) 1.71% and (g) 1.09%.

Differential scanning calorimetry

A complex system can be formed with the addition of hydrocolloids to the starch, and when it is subjected to heating in the presence of water, a suspension of various particles leached from the swollen granules and a network of polymers is formed, which when interacting with each other may have an effect on the thermal behaviour of the blend relative to the native starch [10]. Thus, gelatinisation phenomenon was studied by differential scanning calorimetry, and the curves obtained are shown in Fig. 2.
Fig. 2

DSC curves of native common vetch starch (a) and its mixture with: b carrageenan, c carboxymethylcellulose (CMC), d guar, e jatahy, f pectin and g xanthan

An endothermic process occurs when the starch is heated in excess of water, as observed by the DSC curves. This results in a colloidal dispersion, causing the loss of birefringence and the crystallinity of the starch, due to the unfolding of the double helices of amylopectin [33]. With the addition of hydrocolloids, small displacements of the thermal event occurred due to the interaction of these compounds with the starch, which can be better evaluated through the data extracted from the curves and that are presented in Table 2.
Table 2

DSC curves to the gelatinisation process for common vetch native starch (a) a and its mixture with: (b) carrageenan, (c) carboxymethylcellulose (CMC), (d) guar, (e) jatahy, (f) pectin and (g) xanthan


DSC results




ΔHgel/J g−1


64.55 ± 0.03b

69.81 ± 0.14c

74.46 ± 0.06d

9.59 ± 0.21b


65.51 ± 0.10a

70.58 ± 0.09a

75.49 ± 0.24bc

9.39 ± 0.27b


63.78 ± 0.05c

70.12 ± 0.13bc

75.51 ± 0.18b

7.51 ± 0.03c


63.18 ± 0.14d

70.15 ± 0.04bc

75.08 ± 0.13c

11.03 ± 0.08a


64.04 ± 0.14c

69.82 ± 0.15c

76.29 ± 0.03a

11.06 ± 0.42a


64.49 ± 0.09b

70.24 ± 0.08b

75.31 ± 0.19bc

6.43 ± 0.37d


64.50 ± 0.36b

69.67 ± 0.24c

75.11 ± 0.49c

6.74 ± 0.14d

(*) To “onset” initial temperature, Tp peak temperature, Tc “endset” conclusion temperature, ΔHgel gelatinisation enthalpy. Values presented as mean values ± standard deviation after analysing in triplicate. Values followed by the same letter in the same column are not significantly different by Tukey’s test (p < 0.05)

Native sample showed similar values to the previous study [16]. A decrease in To was observed, differing statistically (p < 0.05) from the native sample, with addition of CMC, guar and jatahy gums. This may be correlated with the decrease in pasting temperature after addition of hydrocolloids observed by viscoamilographic analysis (Table 3).
Table 3

Pasting properties for the native common vetch starch (a) and mixed with: (b) carrageenan, (c) carboxymethylcellulose, (d) guar, (e) jatahy, (f) pectin and (g) xanthan


Pasting temperature/°C

Viscosity peak/mPa s

Setback/mPa s

Breakdown/mPa s

Final viscosity/mPa s

Peak time/s


75.58 ± 0.08a

793.72 ± 0.58g

431.91 ± 0.22g

24.83 ± 0.70f

1200.76 ± 0.61g

624.30 ± 0.20c


75.65 ± 0.05a

871.53 ± 0.66f

667.98 ± 0.89a

205.69 ± 0.95a

1333.40 ± 0.60f

680.55 ± 0.13a


74.75 ± 0.07ab

1122.07 ± 1.96a

589.68 ± 0.71c

41.53 ± 0.40e

1670.49 ± 0.62a

551.94 ± 0.04g


75.10 ± 0.17a

991.59 ± 0.24d

583.91 ± 0.53d

8.22 ± 0.41g

1567.03 ± 0.71c

628.48 ± 0.48b


75.16 ± 0.25a

972.39 ± 0.50e

492.72 ± 1.26e

64.34 ± 0.48d

1402.44 ± 0.28e

604.27 ± 0.21d


74.82 ± 0.52ab

1108.96 ± 1.60b

603.31 ± 0.37b

92.27 ± 0.47b

1619.28 ± 0.35b

568.79 ± 0.09f


73.94 ± 0.64b

1059.67 ± 0.68c

484.61 ± 0.47f

73.50 ± 0.40c

1470.60 ± 0.56d

584.53 ± 0.35e

(*) mPa s “millipascal-second”, s “second”. Values followed by the same letter in the same column are not significantly different by Tukey’s test (p < 0.05)

CMC, pectin and xanthan favoured a decrease in the energy required for the starch gelatinisation, such as obtained by Adamovicz et al. [27] with interaction between pectin and potato starch, and by Chandanasree et al. [34] for cassava starch with CMC. The addition of carrageenan caused an increase in To, Tp and Tc, without changes of gelatinisation enthalpy in relation to the native sample. The increase in the gelatinisation enthalpy observed after addition of guar and jatahy gums may be related to the reduction of the water mobility provided by the gums, preventing the amylose leaching and consequently the swelling of the granule [35], resulting in small viscosity increases as obtained by rapid visco analysis (RVA). Guar gum is a neutral polysaccharide formed by mannose units linked by 1-4-glycosidic bonds, forming a linear chain, with branching points containing galactose chains joined by 1–6 linkages, and exhibits fast water absorption reducing its availability for granule swelling, as discussed by Ptaszek et al. [36]. In addition, guar gum is not a pure polysaccharide and may have the presence of proteins and fibres, which compete for water, making difficult the starch gelatinisation [10].

An increase in Tc, as observed in this study, was also reported for blends of wheat and potato starches with hydroxypropylmethylcellulose (HPMC), konjac glucomannan and arabic gums, which was attributed to the slower process of gelatinisation with delay in the migration of water into the starch granule caused by the addition of hydrocolloids [17]. Torres et al. [37] observed a slight delay of chestnut starch gelatinisation with the increase in the enthalpy after the addition of guar gum.

Differences that are noted for the results obtained in comparison with other studies involving the starch–hydrocolloid interaction, mainly in relation to gelatinisation, may occur as a function of the starch and the type and concentration of hydrocolloid used [38, 39].

Pasting properties

The starch gelatinisation phenomenon is one of the properties of great industrial interest because it imparts interesting characteristics to the foods according to the moisture and heat content employed, as well as the amylose–amylopectin ratio found in the starch and its interaction with the other components present in the system [37]. During gelatinisation, a transition phase stimulated by heating a starch suspension in excess of water provides swelling of the granule from the internal diffusion of water. Parallel to this, loss of crystallinity and leaching of amylose occurs. Thus, hydrocolloids can be mixed to the starch to form hydrogen bonds with the OH groups of amylose mainly, but also with amylopectin, changing the thermal behaviour and pasting properties [40].

The curves obtained by viscoamylographic analysis of native starch and starch–hydrocolloid mixtures are shown in Fig. 3:
Fig. 3

RVA curves to analyse the pasting properties of native common vetch starch (a) and its mixture with: b carrageenan, c carboxymethylcellulose (CMC), d guar, e jatahy, f pectin and g xanthan

From the RVA curves, it was visualised that the behaviour of native common vetch starch differs from other starches as potato and waxy maize starches [41] and cassava starch [23], by presenting a low breakdown, indicating that the swollen granules do not undergo severe disintegration under shear and heat conditions applied in this analysis. However, with cooling, it was observed that the high tendency for retrogradation favoured obtaining final viscosities higher than the peak viscosities. The data obtained from the RVA curves are presented in Table 3.

Together with the decrease in pasting temperature, it was observed that the addition of hydrocolloids provided an increase in the peak viscosity of the starch suspensions, mainly with the use of CMC (a chemically modified hydrocolloid), pectin and xanthan. Starch extracted from four rice varieties with different levels of amylose also showed increase in peak viscosity when added with xanthan, CMC and guar gums [42]. Navy bean starch presented an increase in the viscosity peak only with guar gum; however, pectin and xanthan promoted a decrease in this parameter [43]. The addition of carrageenan gum had the lowest effect on viscosity as obtained by Shi and BeMiller [8] in corn starch. A good interaction hydrocolloid–amylose favours the increase in the viscosity of the dispersions [17]. Studies by Gularte and Rosell [4] found that gums, due to the hydrophilic nature, are capable of increasing the swelling power of maize and potato starches according to the added concentration of the hydrocolloid, as well as the origin of the starch.

Therefore, when minor effects on starch viscosity were noted (with addition of carrageenan, guar and jatahy gums), this may be related to the inhibition of amylose leaching due to the hydrocolloids have formed a coating film with greater compression of the granules preventing its swelling [18]. This behaviour was reported for sweet potato added of xanthan gum [44].

Nawab et al. [45] reported a decrease in pasting temperature (Tp) of mango kernel starch when added to guar and xanthan gums, probably due to the interactions between hydroxyl groups of hydrocolloids and starch. Shi and BeMiller [8] further claim that these interactions occur via synergism with the amylose molecules, first leached during gelatinisation, and later, just prior to pasting formation, with the amylopectin leached. This slight decrease in pasting temperature was also observed in this study, except with the interaction of common vetch starch and carrageenan gum.

The retrogradation tendency increased for all the samples as well as obtained for anionic tapioca starch added with guar gum [9]. This may occur when a high concentration of gums is added to the starch, causing an increase in the rate of retrogradation due to the thermal thickening and the increase in the effective concentration of amylose and amylopectin in the continuous phase [40].

Starch granule fragility observed by breakdown parameter increased to the samples, except with the addition of guar gum. This behaviour was also noted for waxy corn star mixed with xanthan gum at different concentrations [46] and tapioca starch mixed with xanthan gum [9].

The final viscosity was higher for all samples, as observed for rice starch with HPMC, guar and xanthan gums [20], and according by [47], that attributed this increase to the associations between starch and hydrocolloid molecules.

X-ray powder diffraction

Amylose and amylopectin are responsible for the amorphous and crystalline regions, respectively, of the starch granules [48]. Thus, starch is considered a semi-crystalline polymer, and this relative crystallinity can be quantified by X-ray diffractometry.

Diffractograms obtained for all the samples are shown in Fig. 4. Common vetch is a leguminous and as expected, the diffraction pattern of the starch isolated from its seeds was classified as C-type, with peaks identified at 15°, 17° and 23° at 2θ, as obtained in a previous study [16], and this pattern was not altered after its mixture with hydrocolloids.
Fig. 4

X-ray diffractograms of native common vetch starch (a) and its mixture with: b carrageenan, c carboxymethylcellulose (CMC), d guar, e jatahy, f pectin and g xanthan

From diffractograms, the degree of relative crystallinity was calculated for all the samples obtaining the following results: (a) 25.21 ± 0.33%c; (b) 29.52 ± 0.62%ab; (c) 30.30 ± 0.14%a; (d) 26.29 ± 0.15%c; (e) 29.32 ± 0.99%ab; (f) 29.32 ± 0.07%ab; (g) 28.61 ± 0.68b. It was observed that the addition of hydrocolloids to the common vetch starch promoted an increase in the relative crystallinity of the samples, except for the interaction with guar gum, which did not differ statistically to the native starch by 5% significance by Tukey’s test. This increase may be related to the peak overlap of the gums which presented peaks at about 20° (2θ) according studies found in the literature [49, 50].

Alberton et al. [29] concluded that the degree of relative crystallinity of cassava starch was not significantly affected by the addition of 1% hydrocolloids (pectin, CMC, jatahy and xanthan gums). In another study, the relative crystallinity of commercial potato starch decreased when pectin was added [27].


Within the experimental conditions of this study, it was possible to observe an increase in the thermal stability after addition of hydrocolloids to the common vetch starch by thermogravimetry. A decrease in onset (DSC analysis) and pasting temperature (RVA) was obtained with addition of the hydrocolloids, except with carrageenan gum. Differences in the gelatinisation enthalpy occurred as a function of the hydrocolloid used. An increase in peak and final viscosities were achieved, mainly with the use of CMC and pectin. An increase in the degree of relative crystallinity was observed, except with the addition of guar gum. The diffraction pattern, C-type, was not modified after mixing with hydrocolloids.

Thus, promising results were obtained for the use of common vetch as a non-conventional starch source, which after the interaction with different hydrocolloids showed improvements on its technological properties, making interesting future studies of possible applications for food industry.



The authors would like to thank the Brazilian organisation CAPES and CNPq for the financial support and also to thank C-LABMU (UEPG) for help in the analysis.


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Copyright information

© Akadémiai Kiadó, Budapest, Hungary 2018

Authors and Affiliations

  • Camila Delinski Bet
    • 1
  • Cristina Soltovski de Oliveira
    • 1
  • Cleoci Beninca
    • 1
  • Tiago André Denck Colman
    • 2
  • Luiz Gustavo Lacerda
    • 1
  • Egon Schnitzler
    • 1
  1. 1.State University of Ponta GrossaPonta GrossaBrazil
  2. 2.Federal University of Grande DouradosDouradosBrazil

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