Journal of Food Engineering 92 (2009) 448453

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Improved barrier and mechanical properties of novel hydroxypropyl methylcellulose edible lms with chitosan/tripolyphosphate nanoparticles
Mrcia R. de Moura a,d, Fauze A. Aouada a,d, Roberto J. Avena-Bustillos b, Tara H. McHugh b, John M. Krochta c, Luiz H.C. Mattoso d,*
a

Chemistry Department, UFSCar, So Carlos, SP, Brazil Agricultural Research Service, Western Regional Research Center, Albany, CA, United States c Department of Food Science and Technology, University of California, Davis, United States d National Nanotechnology Laboratory for Agriculture, EMBRAPA-CNPDIA, So Carlos, SP, Brazil
b

a r t i c l e

i n f o

a b s t r a c t
Chitosan/tripolyphosphate nanoparticles were prepared and incorporated in hydroxypropyl methylcellulose (HPMC) lms. FT-IR and transmission electron microscopy (TEM) analyses of the nanoparticles, mechanical properties, water vapor permeability, thermal stability, scanning electron microscopy (SEM) of the lms were analyzed. Incorporation of chitosan nanoparticles in the lms improved their mechanical and lm barrier properties signicantly. The chitosan nanoparticles tend to occupy the empty spaces in the pores of the HPMC matrix, increasing the collapse of the pores and thereby improving lm tensile properties and water vapor permeability. The thermal stability of the lms increased with addition of nanoparticles. This study is the rst to investigate the use of CS-TPP nanoparticles for the purpose of strengthening HPMC lms. 2008 Elsevier Ltd. All rights reserved.

Article history: Received 20 June 2008 Received in revised form 15 December 2008 Accepted 18 December 2008 Available online 30 December 2008 Keywords: Chitosan nanoparticles Sodium tripolyphosphate Hydroxypropyl methylcellulose Edible lms

1. Introduction Nanotechnology research is entirely multidisciplinary and the results of such research can be applied very quickly to improve current products (Weitz et al., 2008; Clapper et al., 2008; Kowalczyk and Holyst, 2008). Increasing consumer demands for highquality and microbiologically safer foods, together with longer product shelf life, are continuously forcing researchers and the industry to develop new food preservative strategies. To respond to this latter demand, the food industry makes use of several biodegradable polymer-based edible lms from food-grade proteins, polysaccharides, and lipids that could potentially serve as edible packaging materials (Olivas and Barbosa-Canovas, 2008; Sothornvit et al., 2007). For the past 10 years, many research programs have focused on developing more and more sophisticated edible lms and coatings (Denavi et al., 2009; Sebti et al., 2007). Among them, polysaccharide polymers such as hydroxypropyl methylcellulose (HPMC) and chitosan (CS) have been particularly studied (HernndezMuoz et al., 2008; Prez et al., 2008; Dogan and McHugh, 2007). In the study by No et al. (2007) were discussed the applications of chitosan for improvement of quality and shelf life of vari-

* Corresponding author. Tel.: +55 16 2107 2800; fax: +55 16 2107 2902. E-mail address: mattoso@cnpdia.embrapa.br (L.H.C. Mattoso). 0260-8774/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2008.12.015

ous foods from agriculture, poultry, and seafood origin were focused. However, the hydrophilic nature of such packaging materials, which produces a loss of barrier properties or even a solubilization into foods with high-water activities, prevents their industrial applications (Bertuzzi et al., 2007). Villalobos et al. (2006) reported the syntheses of lms with HPMC, sorbitan monostearate, and sucrose palmitate. The water vapor permeability of these lms was minimal with a hydrocolloid/surfactant ratio. Elsabee et al. (2008) reported the syntheses of lms formulated with polypropylene (PP) and CS/pectin. Chitosan forms complex compounds with pectin and this property was used to build up a stable multilayered structure on the PP lm surface, to produce a much better antimicrobial lm which can be used to fabricate excellent packaging materials for post-harvest crop protection. Preparation methods by ionically cross-linking cationic chitosan with specic polyanions were particularly successful as, aside from its complexation with negatively charged polymers, CS has the ability to gel spontaneously on contact with multivalent polyanions due to the formation of inter- and intramolecular cross-linkage mediated by these polyanions. Among some polyanions investigated, tripolyphosphate (TPP) is the most popular because of its non-toxic property and quick gelling ability. The CS-TPP nano system exhibits some attractive features which render them promising carriers for the delivery of macromolecules (Gan et al., 2005). The application of these nanoparticles in edible lms is very promising due to the food-grade properties

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of both components. Hydroxypropyl methylcellulose lms with CS-TPP nanoparticles could then be a potential material for food packaging applications to extent the shelf life of foods and products. Research and development of bio-nanocomposite materials for food applications, such as packaging and other food contact surfaces is expected to grow in the next decade with the advent of new polymeric materials and composites with inorganic nanoparticles. The application of nanocomposites promises to expand the use of edible and biodegradable lms (Lagarn et al., 2005; Sinha Ray and Bousmina,2005; Sorrentino et al., 2007). Some works (Orts et al., 2005; Okada and Usuki, 1995) has shown that, at relatively low nanoparticle loadings, polymer composites are reinforced because chains within these nanocomposites are restricted to conned domains between sheets of the lms. For instance, the combination of chain connement, alignment of nanoparticles (i.e., nanostructure) and strong surface interaction, results in improvements in mechanical properties, as well as in decreases in gas and liquid permeability. In this paper, chitosan/tripolyphosphate nanoparticles were prepared and incorporated in hydroxypropyl methylcellulose lms. FT-IR and transmission electron microscopy (TEM) analyses of the nanoparticles, mechanical properties, water vapor permeability, thermal stability, scanning electron microscopy (SEM) of the lms were analyzed. The aim of the present study was to investigate the effect of nanoparticles with chitosan on the HPMC lms mechanical and water vapor permeability properties. 2. Materials and methods 2.1. Materials Hydroxypropyl methylcellulose (Methocel E15) was obtained from Dow Chemical Co. (Midland, MI, USA). Chitosan (MW 71.3 kDa, degree of desacetylation 94%) was purchased from Polymar Cincia e Nutrio S/A (Fortaleza, Brazil). Sodium tripolyphosphate was purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). 2.2. Nanoparticles and lm preparation 2.2.1. Preparation of chitosan-tripolyphosphate (CS-TPP) nanoparticles The CS-TPP nanoparticles were obtained according to the procedure rst reported by Calvo et al. (1997), based on the ionic gelation of CS with TPP anions. Chitosan was dissolved in acetic acid solution at concentrations of 3.00 and 4.41 mg mL1. The concentration of acetic acid in aqueous solution was, in all cases, 1.5 times that of chitosan. Under magnetic stirring at room temperature, 28 mL sodium TPP aqueous solution with concentrations of 1.2 and 2.1 mg mL1 were added into 70 mL chitosan solution (Table 1). The preparations were mixed with a homogenizer (Polytron PT 3000 Brinkmann) at 6000 rpm with continuous addition of TPP solution rate of 1 mL min1. The zone of opalescent suspension was further examined as nanoparticles. Table 1 shows the nal concentrations of TPP and CS after different syntheses processes.

2.2.2. FT-IR analysis FT-IR spectra of CS, TPP and CS/TPP nanoparticles were taken with a Paragon 1000 PerkinElmer Spectrum (PerkinElmer Life and Analytical Sciences, Inc., Waltham, MA USA) in the range from 4000 to 400 cm1. The FT-IR spectra were used to determine the existence of TPP and CS in nanoparticles. The CS-TPP nanoparticles were frozen by liquid nitrogen and lyophilized for 12 h, to obtain dried CS-TPP nanoparticles. Powdered samples were prepared using KBr to form pellets. 2.2.3. Particle size distribution The particle size distribution of solutions were measured in a Horiba LA 900 laser light scattering particle size analyzer (Horiba Instruments Inc., Irvine, CA, USA) using laser diffraction to measure particle size of suspensions. All analysis was performed by triplicate. 2.2.4. Transmission electron microscopy (TEM) A Jeol 100 C (Jeol Ltd., Tokyo, Japan) transmission electron microscope (TEM) was used to observe the morphology of the nanoparticles. CS-TPP nanoparticles solutions were sonicated for 1 min to produce better particle dispersion and to prevent nanoparticle agglomeration on the copper grid. One drop of the nanoparticle solution was spread onto a carbon-coated copper grid which was then dried at room temperature for TEM analysis. 2.2.5. Preparation of solutions for lm casting The HPMC solution (control lm) was obtained by dissolving 3.0 g of HPMC in 100 mL of distilled water under magnetic stirring for 12 h. The HPMC: water ratio in all lm-forming solutions was 3:97 to study the effect of particle size and CS-TPP concentration in the HPMC lm matrix. The HPMC lms with CS-TPP nanoparticles were obtained by addition of 3.0 g of HPMC in 100 mL of nanoparticle solution (recently synthesized) under magnetic stirring for 12 h. After the solutions were prepared, the asks were kept closed during 6 h to prevent microbubble formation in the lms. The solutions were then poured in a glass plate (30 30 cm) covered with Mylar (Polyester lm, DuPont, Hopewell, Va., USA) for lm casting preparation. The solutions were cast at a wet thickness of 0.5 mm onto plates using casting bars and the plates were placed on a leveled surface at room temperature and let dry for 24 h. After drying, the lms were removed and conditioned in sealed plastic bags stored at room temperature. 2.3. Film characterization 2.3.1. Film thickness Film thickness was measured using a model 7326, digital micrometer (Mitutoyo Manufacturing, Tokio, Japan) at 5 random positions around the lm. The mean values were used to calculate water vapor permeability and mechanical properties. 2.3.2. Mechanical properties The lms were cut to have rectangular dimensions in accordance to ASTM (1997). The lms were conditioned at 30% RH and 23 C for 48 h before measurements. A Model 1122 Instron Universal Testing Machine (Instron Corp., Canton, MA, USA) was used to determine the maximum tensile strength (TS), maximum percentage elongation at break (%), and elastic modulus (or Youngs modulus). Films were stretched using a speed of 50 mm min1. Tensile properties were calculated from the plot of stress (tensile force/initial cross-sectional area) versus strain (extension as a fraction of the original length). The mechanical properties were analyzed as a function of CS and TPP concentrations.

Table 1 Inuence of chitosan and TPP concentration on the nanoparticle size. CS concentration (mg mL1) 2.14 2.14 3.15 TPP concentration (mg mL1) 0.3 0.6 0.6 Particle size (nm) 85 110 221

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2.3.3. Water vapor permeability (WVP) Water vapor permeability was determined by modication of the ASTM E96-92 gravimetric method to determine the relative humidity (RH) at the lm underside according to McHugh et al. (1993). Five lms were cast from each treatment, onto 15.5 cm internal diameter Teon plates. After drying, one sample without defects was cut from each lm. Distilled water (6 mL) was dispensed into at-bottom Plexiglas cups with wide rims. The lm was sealed to the cup base with a ring using 4 screws symmetrically located around the cup circumference. The cups were placed in temperature-controlled cabinets at 25 C, containing fans and held at 0% RH using anhydrous calcium sulphate (W.A. Hammond Drierite Co., Xenia, OH, USA). Weights were taken periodically after steady state was achieved and used to calculate the % RH at the lm underside and the resulting WVP. Water vapor permeability was calculated using the following relation:

(C)

Transmittance

(B) (A)

4000

3000

2000
-1

1000

Wavenumber (cm )
Fig. 2. Infrared spectra: (A) chitosan; (B) TPP; (C) CS-TPP nanoparticle.

WVTR WVP y p2 p3

where WVTR was obtained from the slope of the weight loss rate through the lm surface and p2 was the water vapor partial pressure on the lm underside calculated according to McHugh et al. (1993) while p3 was water vapor partial pressure at the lm underside and y was the average lm thickness. Water vapor permeability of each lm was measured as the mean and standard deviations of 5 replications. 2.3.4. Thermal analysis The thermal degradation of lms was analyzed in a thermogravimetric analyzer TGA-Q500 (TA Instruments, New Castle, DE, USA). The experiments were performed in the temperature range 20500 C at a heating rate of 10 C min1. The nitrogen ow was maintained at 60 cm3 min1. The analysis was performed in triplicate. 2.3.5. Scanning electron microscopy (SEM) A Hitachi S-4700 scanning electron microscope (Hitachi, Toronto, Ontario, Canada) was used to study the morphology of nanoparticles and lms. The samples were deposited onto aluminum specimen stubs using double-stick carbon tabs (Ted Pella Inc., Redding, CA, USA) and coated with gold/palladium on an ion sputter coated (Denton Vacuum Inc., Moorestown, NJ, USA) for 45 s at 20 mA. All samples were examined using an accelerating beam at a voltage of 1.5 kV. Magnications of 40,000; and 60,000 were used.

2.3.6. Statistical analysis Analysis of variance (ANOVA) was applied using Minitab 14.2 (Minitab Inc., State College, PA, USA) to determine signicance of differences between means. 3. Results and discussion 3.1. FT-IR analysis The molecular structures of chitosan and TPP are presented in Fig. 1. The formation of nanoparticles is conrmed with the FT-IR spectrum showed in Fig. 2. In chitosan-TPP nanoparticles (C) the peak of 3430 cm1 becomes wider, indicating that hydrogen bonding is enhanced (Jia-hui et al., 1999). The peak at 1530 cm1 in the spectrum of the nanoparticles is indicative of interaction between NH 3 groups of chitosan and phosphate groups of TPP. As can be seen from the IR spectrum of chitosan nanoparticles, the peak indicating P@O stretching at 1219 cm1 appear. The appearing of these peaks is an indication of nanoparticle formation. 3.2. Particle size distribution Table 1 shows the inuence of CS and TPP nal concentrations on the size values of nanoparticles. A gradual increase in the particle size was observed with increase in the CS and TPP concentration. This trend may be explained by the fact that a higher weight CS is less soluble resulting in an increase in particle diameter or even aggregation. Nanoparticles with lowest particle size (85 nm) were obtained when the lowest CS and TPP concentration was used. The effect of CS/TPP concentration on particle size was previously studied. Calvo et al. (1997) reported that the particle size is dependent on both CS and TPP concentrations. Gan et al. (2005) further explored the difference between particles associated with bovine serum albumin (BSA) protein and particle without BSA. BSA loading by the incorporation method signicantly increased particle size in comparison to chitosan-TPP particles (without BSA); this fact occurs possibly due to the larger molecular weight and size of the protein molecule. All resulting lms were transparent, exible and homogeneous. Their surfaces were smooth without pores and cracks. Flexible, transparent and surface smooth lms were obtained after drying the lm-forming solutions containing chitosan and CS-TPP nanoparticles.

Fig. 1. Molecular structure of chitosan (a) and TPP (b).

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70 a 60 b

Table 3 Effect of particle size on WVP and % RH at lm underside. Particle size (nm) (No nanoparticle) 85 110 221 WVP (g mm K1 Pa1 h1 m2) 0.794 0.03 0.331 0.06a 0.451 0.05a 0.587 0.05b
c

RH at lm underside (%) 78.2 0.7NS 79.6 0.3 79.4 0.5 79.5 0.9

Tensile Strength (MPa)

50 40 30 20 10 0 c c

a,b,c Different letters within a column indicated signicant difference at P < 0.05. NS, Not signicant differences between lms.

erties of the HPMC lms, clearly demonstrating the reinforcing effects of the nanoparticles on the lms. 3.4. Water vapor permeability

85 nm

110 nm

221 nm

only HPMC without nanoparticle

HPMC films
Fig. 3. Effect of nanoparticles on tensile strength of HPMC lms. Columns show the means and error bars indicate the standard deviations. Different letters within a column indicated signicant difference at P < 0.05.

3.3. Tensile properties of lms Fig. 3 shows the effect of CS/TPP nanoparticles on tensile strength of HPMC lms. There were signicant differences in tensile strength, demonstrating that the incorporation of nanoparticles modied the resistance of the lm. The tensile strength of the HPMC matrix without nanoparticle was 28.3 1.0 MPa increasing to a maximum value of 62.6 1.0 MPa when the particle size was reduced to a minimum value of 85 nm. Addition of nanoparticles resulted in a stiffer material conrming the reinforcing effect of the nanoparticle in the polymeric matrix, which is consistent with previously results reported (Pereira de Abreu et al., 2007). This study reveals that the increased surface area of smaller size CS-TPP nanoparticles compared to their larger size counterparts increases the interaction of particles with the HPMC matrix. As the water evaporates during lm formation, the CS-TPP/HPMC composite network forms by the establishment of bonding. Hence, the increased surface area reinforces the structure. Second, the bigger size particles may be disturbing the formation of HPMC lm during drying, provoking the fragile structure in the lm. Twoway ANOVA indicated that the addition of nanoparticle to HPMC lms and its decrease of particle size increased tensile strength of the lms. The percentage elongations also change when particle size was decreased as shown in Table 2. The increase of the elongation improved the tenacity of the lms. The elastic modulus did not present signicant variation with different sizes of particles, only increase with addition of the nanoparticles. The elasticity of the lms was preserved with addition of nanoparticles. In these ways, the addition of nanoparticles to HPMC lms results in signicant improvements in lm mechanical properties. Furthermore, the particle size present has a tremendous inuence on the nal prop-

Table 2 Effect of particle size on elastic modulus and % elongation of HPMC lms. Particle size (nm) (No nanoparticle) 85 110 221
a,b,c

Elastic modulus (MPa) 900 34a 1264 65b 1190 93b 1204 64b

Elongation (%) 8.1 0.7b 11.1 1.0c 5.2 2.1a 5.7 1.0a

Different letters within a column indicated signicant difference at P < 0.05.

Many studies in food science have been focused in studying the water barrier properties of various edible lms using proteins, polysaccharides and lipids (Hong and Krochta, 2004; Kim et al., 2003; Prez-Gago and Krochta, 2001). The problem with composite lms in the food industry is the relatively high-water vapor permeability of the edible lms. Permeability in lms is controlled by the diffusivity and solubility of water within the lm matrix. Thus using nanoscience, new forms of tightly linked three dimensional networks can be developed to prevent migration of water in food products. Table 3 shows the values of water vapor permeability of HPMC lm and nanoparticles edible lms at different particles sizes. Validity of WVP comparisons is reliable when the same driving force for water vapor diffusion for each lm formulation is used (McHugh et al., 1993). The relative humidity at the lm underside was not signicant different (79.2 0.6% RH) for the four different lms indicated in Table 3. Two-way ANOVA showed that the presence of nanoparticles in the lms decreased the WVP of the HPMC lms. Important information is that a decrease on the particle size decreased the WVP values. This fact is very important in the nanotechnology eld. WVP of HPMC lm without nanoparticle was 0.794 g mm kPa1 h1 m2. Incorporation of CS/TPP nanoparticles decreased the WVP of HPMC lms to a considerable extent (Table 3). This decrease could be due to the formation of hydrogen bonding between chitosan and the HPMC lm matrix, thereby inuencing water diffusion. The structure of lms containing nanoparticles was more compact inuencing a decrease of water permeability of HPMC lms. Nanoparticles with smaller size induced a greater decrease in WVP values. This fact occurs because small size nanoparticles have more ability in occupying the empty spaces of the porous HPMC lm matrix, what difcult the diffusion of water in the lm. In the work described by Lotti et al. (2008) was observed that nanocomposites lms (HDPE + organophilic treated clay) had a signicant decrease of barrier properties (water and oxygen permeability) in comparison to the pure HDPE lm. This trend was attributed to the formation of water clusters (which increase the penetrant volume) the main transport controlling mechanism could be the diffusion through the polymer. The effectiveness of the reinforcing effect of nanosize microcrystalline cellulose (MCC) llers in HPMC lms was described by Dogan and McHugh (2007). In this study was shown that through the incorporation of llers such as MCC, edible lm mechanical properties can be significantly improved without reducing lm barrier properties, especially water vapor permeability (WVP). Rhim et al. (2006) studied the inuence of the nanostructures on WVP values of the chitosan lms. The WVP value of the chitosan lm was 1.31 0.07 g mm K1 Pa1 h1 m2. Already, WVP of the nanocomposite lms decreased signicantly (P < 0.05) by 2530% depending on the nanoparticles. This is attributed to the presence of ordered dispersed nanoparticle layers with large aspect ratios in the polymer matrix,

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100 80

232 C (A) 271 C (B)

Mass Loss (%)

60 40 20 0

100

200 300 400 Temperature (C)

500

600
Fig. 5. Electron transmission microphotography of CS-TPP nanoparticle corresponds to the 3.00 mg L1 CS and 1.2 mg L1 TPP (85 nm) lm solution.

Fig. 4. TG scans of the HPMC lm (A) and HPMC lm with CS-TPP nanoparticle (110 nm) (B).

what contributes to the water vapor traveling through the lm to follow a tortuous path through the polymer matrix surrounding the nanoparticles, thereby increasing the effective path length for diffusion. 3.5. Thermal degradation Fig. 4 shows the TG scans of HPMC lms and HPMC lms content CS-TPP nanoparticles. As the temperature is increased the mass remains constant until about 232 C for HPMC lm and 271 C for HPMC lm with 110 nm CS/TPP nanoparticles. These temperatures correspond to the beginning of thermal degradation (TD). The thermal degradation temperature was very similar for all lms containing nanoparticles (Table 4). The additions of nanoparticles in HPMC lms modied the thermal characteristic of the lms. Two-way ANOVA showed that the presence of nanoparticles in the lms increased the thermal stability of the HPMC lms. This fact is relevant for practical lm casting. A pilot plant labcoater for edible lm casting operates typically at 100132 C to dry the lm in 12 min (Du et al., 2008). 3.6. Film morphology TEM micrograph of the CS-TPP nanoparticles prepared with a 3.0 mg mL1 of CS and 1.2 mg mL1 of TPP solution shows distinctive spherical-shaped nanoparticles (Fig. 5). These nanoparticle size values are smaller than those obtained by laser diffraction (85 nm), as expected, since TEM analyses were carried out in dried nanoparticles samples, whereas the former used aqueous solution and the nanoparticles were in swollen state. The morphology of the composite HPMC lms containing nanoparticles (221 nm) was analyzed by scanning electron microscopy (SEM). Control lm prepared from a solution containing only 3% w/v of HPMC in water (Fig. 6a), exhibited a high degree of porosity evenly distributed

throughout the lm. Compaction of HPMC lm was observed when chitosan nanoparticles were added as shown in Fig. 6b. This compacting further decreased WVP and increased tensile properties of lms.Analysis of the morphology of lms using SEM is extensively discussed in literature. Xiong et al. (2008) showed that the structures of lms were smooth and compact and lms had several different holes, suggesting that the miscibility and compatibility in

Table 4 Thermo-gravimetric parameters of HPMC lms with nanoparticles. Film HPMC HPMC + nanoparticle HPMC + nanoparticle HPMC + nanoparticle Particle size (nm) No nanoparticle 85 110 221 TD (C) 232 279 271 276

Fig. 6. Scanning electron microphotographies of (a) HPMC lms and (b) HPMC lms with CS-TPP nanoparticles of 221 nm.

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each lm is different. These conclusions are taken from analyses of SEM. 4. Conclusions CS-TPP nanoparticles in HPMC lms improved barrier and mechanical properties. From FT-IR analysis, it is proposed that the ionic gelation of CS with TPP anions resulting in nanoparticle formation. SEM images reveal that HPMC lms become more compact and dense when chitosan nanoparticles were added. There were signicant differences in tensile strength, demonstrating that the incorporation of nanoparticles modied the resistance of the HPMC lm. The thermal degradation temperature was increased in lms containing nanoparticles from 232 C to 271 C. This fact is important because the presence of nanoparticles in the HPMC lms improved the thermo-stability of the lms. The implication of this nding to produce compression-molded sheets indicates that CS-TPP nanoparticles can be extruded into lms for formation of edible or biodegradable lms. These ndings indicate that use of nanotechnology can improve functionality to edible lms for food applications. Acknowledgements The authors are grateful to CNPq (Processes MRM-141694/ 2005-0, FAA- 141695/2005-6 and PQ 307.935/2004-3), Embrapa-Brazil (Labex Program and MP1 Project). FINEP/LNNA (O1.06.0096.00) and USDA, ARS, WRRC for their nancials supports. References
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