Self-supported Films of Amburana Cearensis Bipolymer as an Alternative for Biodegradable Packaging

Self-supported �lms, prepared from Amburana cearensis gum (GAmb), appear as an alternative for the development of biodegradable packaging as a way to minimize the impacts of non-biodegradable residue discarded in the environment. The �lms were produced using the casting method and the results obtained from the FTIR analysis indicated that the GAmb is mostly constituted by α-L-Arabinofuranose and β-Galactopyranose units. The �lms, (GAmb/Gly) produced with the addition of glycerol (Gly) at concentrations of 10–30%, presented a variation of 8.4–12.7% in moisture content and from 33–49% for solubility water, respectively. The �lms also showed amorphous characteristics and a transmittance below 50%, as well as a maximum elastic modulus of 8.51 Mpa. The results also showed that the complete biodegradability of the �lms occurred after 14 days, corroborating the hypothesis of using GAmb/Gly �lms as an alternative for biodegradable packaging.


Introduction
Natural polymers or biopolymers, as well as polysaccharides, have been used in the development of selfsupported lms, as they are materials made from renewable sources, have a low production cost and may be biodegradable under different conditions [12].
Polysaccharide lms have been an alternative to replace those based on synthetic materials, to reduce the accumulation of non-biodegradable residues discarded in the environment [9].In 2018, approximately 359 million tons of plastic were produced on a global scale, which has sparked several concerns involving the non-biodegradability of these materials in nature, as well as the negative impact on the terrestrial and aquatic ecosystems [24].Thus, polysaccharides, such as cellulose [23], chitosan [31] and starch [2], have to come to be studied and applied to the development of lms for biodegradable food packaging.
The packaging has different functions, such as protecting the food from the external environment, reducing the interaction of the product with factors that lead to its degradation such as contamination by microorganisms, contact with water vapor, oxygen, or with different compounds that may alter the taste of the food, to extend the shelf life of the product [15].
In other cases, packaging can play the role of a semipermeable barrier [26], reducing product contact with water vapor and oxygen.This function is mainly observed in applications that use biopolymer lms on minimally processed products, in the case of ready-made foods such as fruits and vegetables [14], as well as fresh salmon llets [6].However, the preparation of self-supported polysaccharide-based lms is often limited by the emergence of cracks after the process of drying the lms, making them brittle and a little exible [16].Thus, a strategy that has been employed to improve the mechanical properties of a lm, such as the reduction of its rigidity, has been to add plasticizers [8].
Plasticizers are additives that act by reducing rigidity between the polymer chain molecules, resulting in an increase in the exibility of the material and consequently of the lm [27].Among the plasticizers commonly used, we can mention glycerol (Gly), sorbitol, and polyethylene glycol [18], with the latter being the most used in the preparation of lms obtained from the evaporation of a solvent present from the lm-forming solution, a technique also known as casting, which is done to lms with an excellent visual aspect and attractive mechanical properties [4].
The use of new biopolymers, aiming at the formation of self-supported lms and the development of biodegradable packaging, has attracted the attention of different research groups around the world [29].
In this sense, the species Amburana cearensis (A.cearensis) stands out for presenting in its composition substances with antioxidant, antibacterial, antifungal, and anti-in ammatory properties, mainly due to the presence of phenolic and avonoid compounds in different parts of the species, such as in the methanolic extract of the exudate, stem bark, and seeds [20].Despite all the described properties, until now, no studies have been found regarding the use of the polysaccharide present in the exudate of the A. cearensis species for the development of lms and especially casting lms.This nding indicates the innovative character of this study, which makes use of biopolymer extracted from the exudate of the Amburana cearensis species in the formation of selfsupporting lms developed by the casting method and the results point to its possible use in the development of biodegradable food packaging.

Exudate extraction and GAmb isolation process
Initially, cuts were made in the trunk of A. cearensis; after the exudation process, the exudate was collected and exposed to the sun for two weeks until it had fully solidi ed.Subsequently, the dried exudate was stored to be used in the gum isolation process [22].
Then, the solid exudate was macerated and passed through a 75 µm particle size sieve to remove major impurities and reduce particle size.Afterward, 5 g of exudate was added to 100 ml of distilled water and left to stir (at 1000 rpm) for 1h.At the end of this period, the solution with pH 4.2 was neutralized (pH 7.0) by adding 0.05 M Sodium Hydroxide.Subsequently, 1 g of Sodium Chloride was added to facilitate the precipitation of the polymer in the subsequent step.The resulting solution was stirred at 1000 rpm for 1 h.After that time, 300 mL of Ethylic Alcohol was added to it and, after approximately 1 minute of stirring, this was stopped, so that the precipitation process occurred within 20 minutes.After the precipitation time, the supernatant was removed from the precipitate, leaving only the polymer isolated and the nal yield was calculated to be 86%.Finally, the material was dried at 100°C for 3 h, then macerated, passed through a 75 µm sieve, and stored for subsequent lms preparation.
For the formation of the GAmb lm containing 10% Gly, initially, 0.06 g of glycerol in 30 ml of distilled water was added and the resulting mixture was left under stirring at 900 rpm for 5 minutes.Then, slow addition of 0.6 g of GAmb was made to avoid the formation of agglomerates of the biopolymer during the mixture, which was kept under stirring (900 rpm) for 30 minutes.After that time, the solution was heated to 80°C and, upon reaching this temperature, stirring was continued for another 30 minutes.At the end of 30 minutes, heating was stopped, and stirring continued until the solution cooled to around 50°C.Then the lm-forming solution was homogeneously dispersed in a plastic Petri dish (size 90 x 15 mm) and taken to the oven, where it remained for 20 h at 50°C for total solvent evaporation.Subsequently, the plate was removed from the oven and kept at room temperature (± 22°C in an air-conditioned room) until its complete cooling, when the lm was removed from the plate, which was stored for subsequent characterizations.The other lms prepared at concentrations of 15-30% of glycerol followed the same methodology and for comparison purposes, GAmb lms were also prepared without the addition of glycerol, that is, 0% Gly.

Fourier transform infrared spectroscopy (FTIR)
The characterization of the lms by FTIR was performed with the aid of a Spectrum 100 (PerkinElmer) spectrometer, using the attenuated total re ectance (ATR) mode, in the range of 4000-600cm − 1 , with an accumulation of 16 scans, resolution of 4 cm − 1 and 0.5 cm − 1 range.

X-ray diffraction (XRD) analysis
The X-ray diffractograms of the lms were obtained using Shimadzu equipment, model XRD-6000, with CuKα radiation (λ = 1.5418Å), tube voltage of 40kV and current of 30mA, with a scanning speed of 2°/min and 2θ angle ranging from 5° to 75°.

Biodegradation study
The biodegradability test of the lms was carried out at the Laboratory for Research and Development of New Materials and Sensor Systems (MATSenS) of the Federal University of Piauí -UFPI, and the soil was collected at the Technology Center -CT of UFPI in April 2021, at coordinates 5°3'23"S and 42°47'59"W.
Initially, the collected soil was passed through a sieve with a diameter of 55 cm and an internal mesh of 2.8 mm x 4.5 mm to remove larger stones and make the soil more homogeneous, then the soil was stored for the biodegradation tests.

Biodegradation by mass loss
At rst, the GAmb lms with glycerol were cut in dimensions of 14 mm x 14 mm and the initial mass (W 1 ) of each sample was determined.Then, they were wrapped in polyester fabric to facilitate the removal of the lms after the test.The polyester/sample systems were added, at a depth of 7 cm, in containers containing 200 g of previously collected soil.Subsequently, these containers with soil and the samples were sprinkled with 120 mL of distilled water and kept at room temperature (± 22°C in an acclimatized room).Day after day, the lms were removed from the containers, dried in an oven at 105°C until they reached a constant mass and then their nal mass (W 2 ) was determined, totaling 14 days of analysis.
Finally, all experiments were done in triplicate.Percent biodegradation (%) was calculated as a function of mass loss using Eq. ( 1): 2.6.2.Biodegradation by released CO 2 Samples of the 14mm x 14mm size lms were placed at a depth of 7 cm in containers containing 200 g of the previously collected soil.Subsequently, the soil was sprayed with 120 mL of distilled water, and a transparent polypropylene bottle containing 20 mL of 0.1 M Sodium Hydroxide was attached to this system to capture the CO 2 released by the microbial activity of the lm in the ground.The containers were hermetically closed and kept at room temperature (± 22°C in an air-conditioned room).The same procedure was performed for a soil sample without the presence of any lm, which is called control system or test blank.Day after day, the amount of CO 2 produced by microbial activity in the soil was evaluated, totaling 14 days of analysis.
The quanti cation of CO 2 was obtained by titration of a 0.1 M NaOH solution with a 0.5 M HCl solution, using barium chloride (BaCl 2 ) for precipitation of carbonate ions, in addition to phenolphthalein as an indicator solution.Carbon dioxide levels could be calculated and plotted as a function of incubation time [1].The quanti cation of CO 2 produced in the containers was generated from the spent volume of 0.5 M HCl in the titration of 0.1 M NaOH; therefore, the amount of CO 2 generated in each container was calculated in mg using Eq. ( 2): Where CO 2 = carbon dioxide generated; V B = volume of 0.5 M HCl spent in the blank titration.;V A = volume of 0.5 M HCl spent on sample titration; 50 = factor to transform equivalent in µmol of CO 2 ; f HCl = 0.5 M HCl factor e 0,044 = factor to transform µmol into mg of CO 2 .

Mechanical test
The mechanical tensile properties of the lms were obtained in a TA.XT plus texturometer (Stable Micro Systems, United Kingdom), according to the procedures described by the ASTM D882-99 standard.All tests were performed in triplicate.

Optical property
The UV-Vis spectra of the lms were obtained on a Varian Cary 500 UV-Vis spectrometer in the wavelength range of 200-800nm.

Thickness
The average thickness of each lm was determined using a DIGIMES analog micrometer with a scale from 0 to 1 mm and precision of 0.01 mm, from measurements at fteen different points in each sample.

Moisture content
The measurement of moisture content (M C ) was performed for the lms with glycerol in samples with dimensions of 20 mm x 20 mm; initially, the initial mass of each lm (M i ) was determined, then the lms were dried in an oven for 24h at 105°C.Subsequently, the nal mass of the lms (M f ) was determined and, therefore, the measurements were carried out in triplicate and the average of the values were used in Eq. ( 3) to determine the moisture content.

Water solubility
Initially, the initial mass (M o ) of the lms was determined in the dimensions of 20 mm x 20 mm; then, the lms were immersed in 30 mL of distilled water and stirred on a stirring table, Orbital SL − 180/D, for a period of 24 h at 180 rpm.After this period, the samples were dried in an oven at 105°C for 24h, and nally, the nal mass (M ) of each sample was determined, as well as the water solubility (S W ) of the lms by means of Eq. ( 4), and again, all measurements were performed in triplicate.

Infrared Spectroscopy
The FTIR analyses of the cast lms formed by both GAmb alone and GAmb added glycerol, Fig. 1, allowed the identi cation of the functional groups present in the biopolymer, as well as to evaluate the effect of increasing the concentration of glycerol in the lm.
In Fig. 1, the characteristic vibrational band of hydroxyl groups -O-H was observed at approximately 3400 cm − 1 , while the band corresponding to the vibrational stretch of -C-H, characteristic of methyl and methylene groups, was observed at 2927 cm − 1 .Furthermore, the vibrational bands at 1616 cm − 1 and 1421 cm − 1 can be attributed to the vibrational stretching of symmetric and asymmetric -C = O groups, respectively.Therefore, the 1038 cm − 1 band can be attributed to the vibrational stretch of -C-O-C-of uronic acid [21].Moreover, the FTIR results of GAmb lms indicate that the biopolymer is mainly constituted by α-L-Arabinofuranose and β-Galactopyranose units, Fig. S1 (supplementary material), suggesting that its basic structure is mainly composed of arabinogalactan heteropolysaccharides.
Still in Fig. 1, it is possible to verify that the increase in the concentration of glycerol in the GAmb lms promotes an increase in the intensity of the FTIR bands, which indicates that this behavior may have resulted from hydrogen bonds between the -OH groups of the plasticizer and the biopolymer.

Study of moisture content and water solubility
Figure 2 shows the moisture content and water solubility studies of the GAmb lms prepared with glycerol; this study was not performed for the GAmb lm without the presence of glycerol due to the fragility of the lms.The moisture contents of the lms ranged from 8.4%, for the GAmb/Gly(10) lm, to 12.7%, for the GAmb/Gly(30) lm.Therefore, these values indicated the existence of few water molecules adhered to the molecular chains of the GAmb biopolymer with glycerol, which could guarantee greater durability of the packaged product due to the lower amount of water present in the GAmb lms.
The moisture content found for GAmb lms makes them promising materials for applications in minimally processed food packaging, such as fruits, sh llets, and others, as they have lower moisture contents than those reported in the literature for different lms based on polysaccharides, as in chitosan, pectin and lemongrass essential oil lms, which had a moisture content ranging from 28.78-31.35%[11].
Therefore, lms with lower moisture content tend to have lower interactions between the water molecules in the lms and the packaged product, resulting in greater e ciency in food preservation.
Different explanations can justify the lower moisture content of GAmb lms, one of which can be attributed to the chemical structure of the polysaccharide, where the dense crosslinking between the chains decreased the accessibility of -OH groups by the water molecules [25].
The water solubility values of GAmb/Gly lms ranged from 33-49% (Fig. 2); in addition, considering the hydrophilic nature of GAmb, the lms were not fully dissolved in water, but partially lost their integrity with time.Water solubility results indicated that GAmb/Gly lms have moderate water resistance.

Film diffractograms
Figure 3 shows the XRD patterns of GAmb and GAmb/Gly lms, with the broad peak close to 20°, indicating that the gum has an amorphous characteristic.This amorphous characteristic is also noticed in other gums, such as chitosan and Arabic gum [28].The lms containing glycerol at concentrations of 10-30% showed results similar to those obtained for the GAmb lm without glycerol (GAmb); however, an increase in intensity was observed at the 20° peak for the lms with plasticizer, con rming the presence of glycerol in the biopolymer.Accordingly, the plasticizer facilitated the development of GAmb-based lms without changing the amorphous character of the heteropolysaccharide, even after the addition of higher percentages of glycerol.

Biodegradability
The biodegradation of GAmb lms with glycerol is shown in Fig. 4; thus, GAmb lms showed increasing biodegradation over the fourteen days test and higher percentages of glycerol did not in uence the biodegradation of GAmb, justi ed by the similar behavior of the biodegradation curves as a function of mass loss.
Initially, on the rst three days, the decomposition process of the lms occurred in an accelerated manner for all GAmb lms with glycerol; for example, a mass loss of ≈24% was observed for the GAmb/Gly(20) lm on the rst day, while on the second day this value was ≈48%, and subsequently, on the third day, it was ≈59%.
After the third day, biodegradation continued to increase, but slowly, as the results showed that the mass loss intervals decreased.On the sixth day, the lm had already been ≈81% biodegraded; on the tenth day, it was ≈92%, and by the end of the fourteenth day the lm was practically all biodegraded, showing a mass loss of ≈98%.After 14 days, it was not possible to measure the mass of GAmb/Gly lms.
The CO 2 (mg) values produced in the mineralization process of GAmb lms with 10-30% glycerol are also shown in Fig. 4. Initially, it is observed that the curves of GAmb/Gly lms have similar behavior, even in higher amounts of plasticizer, demonstrating that glycerol did not in uence the mineralization process of GAmb lms, as was observed in biodegradation by loss (a) of mass.
The release of carbon dioxide occurred gradually and increasingly during the 14 days of testing, for example, the lm with 20% Gly had, on the rst day, a release of 0.0440 mg of CO 2 ; on the fth day; 0.286 mg of CO 2 ; on the ninth day, 0.999 mg of CO 2; and, nally, on the fourteenth day, 1.51 mg of CO 2 .In addition, all lms had similar behavior to the lm with 20% glycerol.These results indicate that the biodegradation process of GAmb lms by the release of carbon dioxide (b) was effective and corroborated the biodegradation test by loss of mass.Therefore, the low degree of crystallinity of GAmb may have positively in uenced the rapid biodegradation process of the lms, as well as its ability to retain water molecules.Tables (S1 and S2), with the values of the mass loss and CO 2 release tests, are found in the supplementary material.
Figure 5 also shows the images of GAmb lms with biodegraded glycerol every two days, totaling 14 days.It is observed that the loss of mass occurred gradually, as with the lm with 20% Gly: on the second day, a mass loss of ≈47% was recorded; on the sixth day, ≈81%; on the tenth day, ≈92%; and, on the fourteenth, ≈98%.Therefore, the visual biodegradation analysis of the lms, shown in Fig. 5, corroborates those observed in Fig. 4.These results showed that the development of GAmb-based lms for biodegradable packaging is promising, mainly due to the rapid biodegradability of GAmb lms.These results are interesting, according to the literature, in comparison with polylactic acid lms, a biopolymer widely used in biodegradable packaging, as its complete biodegradation can occur in months [7].

Mechanical properties
Films based on biopolymers must resist the stress encountered during their application to protect and maintain the integrity of the food, and they must also be exible, adapting themselves to possible deformations without breaking [19].Thus, Fig. 6 shows the stress-strain curves of GAmb plus glycerol lms in different percentages, and Table 1 shows the values of the modulus of elasticity, tensile strength, and elongation to rupture obtained in the mechanical test of the lms.
Figure 6 shows a change in the mechanical behavior of GAmb lms, from brittle to ductile, as the percentage of glycerol is increased.From a mechanical point of view, there was a decrease in tensile strength with the increasing percentage of Gly among GAmb lms with 10% Gly (6.41 MPa), 15% Gly (5.43 MPa), 20% Gly (4.70 MPa), 25% Gly (4.16 MPa) and 30% Gly (2.30 MPa).This is due to the glycerol plasticizer, which, when added to the biopolymer, promotes an increase in the free volume between the biopolymer chains, leading to greater chain mobility and lm exibility.Therefore, this behavior, caused by the addition of plasticizer, is important to make the GAmb lm applicable in packaging, as it increases its deformation capacity.
The lm with 10% glycerol is very rigid, that is, not very exible, with an elastic modulus of 8.51 Mpa and elongation to break of 1.26%, which is not interesting for packaging applications due to its rigidity.
However, the other lms had acceptable deformations for packaging.In that case, the lm with 15% Gly had a modulus of 3.44 Mpa and elongation of 2.73%; the one with 20% Gly had a modulus of 1.72 Mpa and elongation 12.20%; the one with 25% Gly had a 0.89 Mpa modulus of elasticity and 16.76% elongation, and the one with 30% Gly had a 0.33 Mpa modulus of elasticity and 21.79% elongation.All values are presented in Table 1 and, in graphic form, in Fig. 6.Therefore, among the lms, those that presented the best mechanical values for packaging purposes are among the lms with 15% and 30% Gly.
According to the data shown in Table 1, the samples containing the plasticizer glycerol showed an increase in the elongation to rupture, indicating that the presence of Gly acted by increasing the deformation capacity of the lms with 15%, 20%, 25% and 30% Gly.

Transmittance determination and visual aspect
The light transmittance spectra of GAmb lms without glycerol (GAmb) and containing Gly at different concentrations are shown in Fig. 7.The values found were displayed in Table 2, at wavelengths ranging from 300nm to 800nm.
The light transmittances of GAmb lms were below 50% (Fig. 7) and, as the percentage of glycerol was increased from 10-30% of Gly, the transmittance values gradually decreased: at 400 nm, it decreased by 22.17-12.50%;at 600 nm, it decreased from 33.25-19.75%;and, at 800 nm, it decreased from 35.72-22.02%,as shown in Table 2.The results showed that GAmb lms became less transparent with the increasing amount of Gly.The reduction in the transmittance values of the lms was possibly due to the presence of the plasticizer, which may have acted as an obstacle to the passage or the scattering of light in the lm matrix [3].
Also, as stated above, the results of GAmb lms with glycerol were better than for other biopolymer lms, such as cassia gum [5], gum arabic [13] and chitosan/shellac [30] which had transmittance greater than 80%.Meanwhile, GAmb lms' transmittance was below 50%, acting as a more e cient barrier against the passage of UV (ultraviolet) and visible light.Although light transmission decreases with increasing Gly concentration, GAmb lms remained transparent.Thus, the results indicated that GAmb lms are interesting for applications in transparent packaging and that glycerol can reduce the effect of UV and visible light on packaged products.Figure 8 shows the images of the Amburana cearensis gum lms and, rst, ripples and cracks are observed in the GAmb lm, indicating its fragility.Furthermore, this fragility can be attributed to the strong intermolecular bonds of the compounds that make up the polysaccharide, providing less mobility to the molecular chains and resulting in a rigid and brittle lm [17].
As explained above, the use of glycerol for the development of GAmb-based lms was important, since this plasticizer, when it remains in the intermolecular spaces of the polymer chains, can promote a

Figures Figure 1 FTIR
Figures

Figure 7 Film
Figure 7

Table 2
Transmittance values of GAmb lms.