- Email: [email protected]
Bionanocomposite films based on polysaccharides from banana peels
Accepted Manuscript Title: Bionanocomposite films based on polysaccharides from banana peels Authors: T´ulio ´Italo S. Oliveira, Morsyleide F. Rosa, Michael J. Ridout, Kathryn Cross, Edy S. Brito, Lorena M.A. Silva, Selma E. Mazzetto, Keith W. Waldron, Henriette M.C. Azeredo PII: DOI: Reference:
S0141-8130(16)31273-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.068 BIOMAC 7234
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
17-8-2016 10-3-2017 12-3-2017
Please cite this article as: T´ulio ´Italo S.Oliveira, Morsyleide F.Rosa, Michael J.Ridout, Kathryn Cross, Edy S.Brito, Lorena M.A.Silva, Selma E.Mazzetto, Keith W.Waldron, Henriette M.C.Azeredo, Bionanocomposite films based on polysaccharides from banana peels, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.068 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bionanocomposite films based on polysaccharides from banana peels
Túlio Ítalo S. Oliveiraa, Morsyleide F. Rosab, Michael J. Ridoutc, Kathryn Crossc, Edy S. Britob, Lorena M. A. Silvab, Selma E. Mazzettoa, Keith W. Waldronc, Henriette M. C. Azeredob,*
aFederal
University of Ceara, Organic and Inorganic Chemistry Department, Campus
Pici, 60440-900, Fortaleza, CE, Brazil; bEmbrapa Tropical Agroindustry, R. Dra. Sara Mesquita, 2270, Pici, 60511-110, Fortaleza, CE, Brazil; cInstitute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK. *Corresponding author, [email protected].
1
Abstract
Pectin and cellulose nanocrystals (CNCs) isolated from banana peels were used to prepare films. The effects of a reinforcing phase (CNCs) and a crosslinker (citric acid, CA) on properties of pectin films were studied. Glycerol-plasticized films were prepared by casting, with different CNC contents (0–10 wt%), with or without CA. Overall tensile properties were improved by intermediate CNC contents (around 5 wt%). The water resistance and water vapor barrier properties were also enhanced by CNC. Evidences were found from Fourier Transform Infrared (FTIR) spectra supporting the occurrence of crosslinking by CA. Additionally, the tensile strength, water resistance and barrier to water vapor were improved by the presence of CA. The
13C
ssNMR spectra indicated that both CA and CNC promoted stiffening of the
polymer chains. Keywords: polysaccharides; biopolymers; bionanocomposites; food packaging.
2
1. Introduction
Concerns about environmental impacts from food packaging waste have motivated development of biodegradable materials, mostly from renewable sources. The global market for biodegradable polymers has increased from 0.4 to 1.3 billion pounds between 2006 and 2013 [1]. Pectins, present in plant primary cell walls and middle lamellae, have a main backbone consisting of -1,4-linked galacturonic acids with variable degree of methoxylation (DM) of their carboxylic acid residues. According to their DM, pectins are classified as high methoxyl pectins (HMP) or low methoxyl pectins (LMP), having DM 50 and DM < 50, respectively [2]. The global annual production of bananas (Musa acuminata, AAA Group) surpassed 100 million tons in 2011, Brazil being the fourth producer [3]. Banana peels (30% of fruit weight) are usually discarded, causing pollution because of their susceptibility to microbial degradation [4]. They have been presented as sources of pectin [5, 6] as well as cellulose [6, 7], which makes their proper exploitation interesting from the perspective of valorization of by-products for non-food applications in a biorefinery context, transforming them into value-added products, decreasing their negative environmental impacts, and enhancing their economic feasibility. Pectins from other sources have been used for development of biodegradable films [8, 9]. However, the use of polysaccharide films for food packaging is still limited due to their poor tensile and barrier properties when compared to those of petroleumderived polymers. The incorporation of nanoreinforcements such as cellulose nanocrystals (CNCs) has been suggested to improve the performance of pectin films 3
[10]. CNCs are needlelike crystals measuring 4-25 nm in diameter and 100-1,000 nm in length [11], often isolated by processes including acid hydrolysis [12]. The most studied effects of CNCs on polymer matrices are on tensile properties, due to the high strength and stiffness of CNCs [13] and to mechanical percolation yielded by CNC hydrogen bonding interactions [14]. Another limitation of polysaccharide-based films is their strong hydrophilic character, which makes them dissolve in contact with water, limiting their applications. Citric acid has been reported to crosslink polysaccharide films by forming covalent di-ester linkages between two of their carboxyl groups and hydroxyl groups of different polysaccharide chains [15, 16]. Carboxyl groups of citric acid form cyclic anhydrides, which react further with hydroxyls from the polysaccharide [17]. The reaction is favored by curing the dried films, typically above 100˚C [16, 18]. Although banana peels have been reported in previous studies as a source of pectins [5, 6, 19, 20] and cellulose nanostructures [7, 21, 22], to our knowledge, this is the first study to propose the development of films from pectin and cellulose nanocrystals isolated from banana peels. Considering that both nanoreinforcing and crosslinking agents have been reported to improve film properties by different mechanisms, this study was conducted to evaluate the combined effects of a nanoreinforcement (CNC) and a crosslinker (citric acid) on tensile, barrier, and water resistance properties of films from banana peel pectin, in order to evaluate if that combination of additives results in films with improved properties when compared with the individual use of each additive.
4
2. Materials and Methods
2.1. Extraction and characterization of pectin from banana peels Banana peels at ripening stage 6 (fully ripe bananas) were collected from Cavendish bananas (Musa AAA group, Cavendish subgroup) purchased in the local market (Norwich, UK). The peels were immersed in Na2S2O5 solution (1% w/v) for 24 h, oven-dried at 60°C for 24 h, and milled to 0.5 mm in a Retsch Brinkmann ZM-1 centrifugal grinding mill (Retsch GmbH, Haan, Germany). The alcohol insoluble residue (AIR) preparation was based on the method by Waldron & Selvendran [23], with some modifications. 100 g of milled peels were washed three times with ethanol to remove alcohol soluble phenolics (firstly with 600 mL boiling 70 v% ethanol for 5 min, then with 600 mL boiling absolute ethanol for 5 min, the third time with 600 mL absolute ethanol for 5 min), then washed in 200 mL acetone. Between washings, the material was filtered through a 10 µm nylon mesh. The AIR was air dried at room temperature, and used thereafter for extraction of both pectin and cellulose nanocrystals. The pectin was extracted with a citric acid solution at pH 2.0 (AIR: solution ratio, 1:20, w/v) at 87°C for 160 min under stirring (150 rpm), and centrifuged at 1,850 g for 30 min at 10°C in a Beckman JS-4.2 centrifuge (Beckman Coulter, Brea, USA). The supernatant was vacuum filtered, had its pH adjusted to 3.5 (minimum pectin solubility) with KOH, and was added with absolute ethanol (1:1, v/v). The suspension was stirred for 30 min and left to precipitate at 4°C for 2 h. After centrifugation (15 min, 4°C, 2,520 g, in the same Beckman JS-4.2 centrifuge), the pellet was collected, 5
washed with ethanol 70 v%, centrifuged again (20 min, 4°C, 2,520 g), and dried at room temperature. It was then stirred in distilled water with a Ystral X10/25 homogenizer (Ballrechtar-Dottingen, Germany) for 20 min, had its pH adjusted to 7, and was again dried and milled by using a basic mill (A10, IKA GmbH, Germany). The DM and the galacturonic acid content (GAC) of the pectin were determined from Fourier Transform Infrared (FTIR) spectra collected in the frequency range of 4000– 800 cm-1 (128 scans at 2 cm−1 resolution) on a Nicolet Magna-IR 860 FTIR spectrometer (Thermo Nicolet, Madison, WI, USA). Samples were placed on a GoldenGate diamond ATR accessory (Specac, Orpington, Kent), the empty crystal used as reference. The DM was determined as described elsewhere [24], with some modifications. FTIR spectra were firstly obtained from pectin standards with known DM values (31%, 67%, and 89%) from Sigma (Steinheim, Germany) and four additional standards obtained by blending the initial standards (for DM values of 40, 49, 60, and 78). The DM determination was based on the band areas at 1700-1750 cm-1 (methyl esterified uronic acids, EUA) and 1600-1630 cm-1 (free uronic acids, FUA), calculated by using multiple peak fit function/Gaussian fitting on Origin Pro 9 (OriginLab, Northampton, USA). The calibration curve (Equation 1), obtained by plotting DM of the standards against the ratio between EUA peak area (AEUA) over the sum of EUA and FUA peak areas (AEUA + AFUA), was used to determine the DM of banana peel pectin.
DM (%) 126.3
AEUA 2.493 AEUA AFUA
( R 2 0.974)
(1)
6
The GAC determination was modified from a previously described method [25]. A set of
10 calibration galacturonic acid standards was prepared by blending
polygalacturonic acid (95%, 81325, Fluka) with cellulose (Sigmacell type 20, Sigma) to several final GAC values (0 to 90 wt%). Peak areas were measured as the total carbonyl peak area above the baseline between 1840 cm -1 and 1550 cm-1 (A15501840).
The calibration curve (Equation 2), obtained by plotting GAC against A1550-1840,
was used to determine GAC of banana peel pectin. GAC (%) 0.7934 A15501840 - 0.1985
( R 2 0.951)
(2)
2.2. Extraction and characterization of cellulose nanocrystals (CNC) Acetosolv pulping was conducted by suspending AIR in a mixture of 93 wt% acetic acid and 0.3 wt% HCl in distilled water, in a 1:10 ratio (w/v, AIR:solution), in a flat bottom flask with reflux at 115 °C under constant stirring (150 rpm) for 3 h. The mixture was filtered through a 28 μm filter paper, resulting in a lignin-rich black liquor and the acetosolv pulp (partly delignified fibers). The acetosolv pulp was rinsed with hot (80°C) acetic acid (99.7%) until the washed liquid was colorless, then washed with distilled water until pH 6.5, and finally oven-dried at 45°C until constant weight. The acetosolv pulp was bleached in two steps. The first one was conducted in an aqueous solution containing H2O2 6% (v/v) and NaOH 4% (w/v) in a beaker at 70°C in a ratio of 1:90 (pulp:solution, w/v) with constant stirring (150 rpm) for 3 h. Then, after being washed with distilled water to pH 7 and oven dried at 60°C for 24h, the pulp was immersed in KOH 6% (w/v) in a 1:20 ratio (w/v, pulp:solution) at 80°C with constant stirring (150 rpm) for 90 min. 7
The acid hydrolysis was then conducted as described previously [26], with some modifications. The bleached acetosolv pulp was stirred (200 rpm) in a 30 v% H2SO4 solution at 45°C, in a 1:20 ratio (w/v, pulp: solution) for 150 min. Immediately following hydrolysis, the suspension was diluted 10-fold with deionized water to quench the reaction, then submitted to three cycles including centrifugation (9,800 g for 15 min at 20°C, in an Avanti J-25 centrifuge, Beckman Coulter, Brea, USA), dilution 10-fold with deionized water, and sonication for 2 min in a probe Branson sonicator (Digital Sonifier 250, Branson Ultrasonics, Danbury, USA) at 20 kHz. After those centrifugation-dilution-sonication cycles, the CNC suspension was dialysed against deionized water for 48 h with four changes of water. The final concentration of the CNC suspension was 0.22 g/100 mL. The zeta-potential (ζ) of CNCs was measured in triplicate using a Zetasizer Nano ZS 3000 (Malvern Instruments Ltd., Worcestershire, UK) for CNC suspensions diluted to 0.1 wt% with deionized water. For the transmission electron microscopy (TEM) images, previously sonicated CNC suspensions were placed onto Formvar/carbon coated grids and negatively stained with 2% uranyl acetate. The samples were imaged using a Tecnai 20 Transmission Electron Microscope (FEI, Hillsboro, USA), with an acceleration voltage of 200 kV, and a magnification of 57500 x. The CNC dimensions (length and diameter) were taken as averages of 100 measurements taken by using the scale bar embedded in the image. 2.3. Film preparation
8
Ten films were prepared, with different CNC contents (0, 2.5, 5, 7.5, and 10 wt% on a dry bionanocomposite basis), with or without citric acid (20 wt% on a dry bionanocomposite basis, as defined from preliminary tests), hereafter referred to as CNC-X-CA-Y, X and Y indicating the percent CNC and citric acid (CA) contents, respectively. For each film, the CNC dispersion (in a volume determined according to its final intended concentration) was collected and added with 4.5 g pectin, 1.35 g glycerol (added as a plasticizer), citric acid, and distilled water, whose volume was calculated to provide a solid content of 2.5 g/100 mL. The film forming dispersion was homogenized in an X10/25 homogenizer (Ystral, Ballrechtar-Dottingen, Germany) for 30 min. Air bubbles were removed under vacuum, and the films were cast on petri dishes to a dry thickness of 0.10 mm, and left to dry at 40˚C in a fan oven (Memmert, Schwabach, Germany) for 16 h. The films added with CA were then cured at 150˚C for 10 minutes using the same fan oven. The dried films were detached from the petri dishes and conditioned (50% RH, 24˚C) in an environmental chamber (Weiss Gallenkamp, Loughborough, UK) for 40 h before the analyses. 2.4. Characterization of films The water vapor permeability (WVP) determination was modified from the ASTM method E96-05 [27] for five samples. The test films had their thicknesses measured (micrometer screw gauge, Moore & Wright, Sheffield, UK) to the nearest 0.01 mm at six random locations, and were sealed as patches onto acrylic permeation cells (2.4 cm in diameter and 1 cm in height) containing 2 mL distilled water. The cells were placed in a desiccator connected to tubes providing a steady flow of dried air (less 9
than 1% RH) from a Balston 75-60 air drier at 24 °C, and were weighed eight times over a 24-h period. For each sample, measurements were taken in at least five replicates. The insoluble matter was determined in quadruplicate on 2 cm x 2 cm film pieces, according to a previously proposed method [28], with modifications. Previously dried and weighed samples were immersed in 50 mL distilled water for 6 h at 25˚C under stirring (150 rpm). The dry weight of the remaining film pieces was obtained after filtration on previously dried and weighed filter paper, and was used to calculate insoluble matter as a percentage of the initial dry weight. The dry weights were determined after drying at 103˚C for 24 h using a fan oven (Memmert, Schwabach, Germany). Tensile tests were conducted (with at least five replicates) on 8 mm x 50 mm specimens on a Texture Analyzer TA.XT Plus (Stable Micro Systems, Godalming, UK) equipped with A/TG Tensile Grips and a 5 kg load cell. The sample thicknesses were determined with a micrometer to the nearest 0.01 mm at 6 random locations. The initial grip separation and crosshead speed were set to 40 mm and 1 mm/s, respectively. Film opacity was determined (in quadruplicate) as described elsewhere [29]. Film strips (1 cm x 5 cm) were placed on the internal side of a Varian Cary 50 UV–vis spectrophotometer
cell
(Agilent
Technologies,
Santa
Clara,
CA,
USA),
perpendicularly to the light beam. The absorbance spectrum (400–800 nm) of film samples was recorded, and opacity was defined as the area under the recorded
10
curve (estimated by the linear trapezoidal rule) and expressed as absorbance units x nanometers (wavelength) per millimeter (film thickness) (A.nm.mm -1). The significance of differences between films without and with CA for tensile properties, insoluble matter, and WVP was established by paired t tests for each response, considering all replicates for all CNC levels. Fourier Transform infrared (FTIR) spectra (128 scans at 2 cm −1 resolution for a spectral range from 4000-800 cm−1) were recorded using a Nicolet Magna-IR 860 FTIR spectrometer (Thermo Nicolet, Madison, WI, USA). Samples were placed on a GoldenGate diamond ATR accessory (Specac, Orpington, Kent). The empty crystal was used as reference. The crosslinking occurrence was assessed by two comparisons between corresponding films with and without CA: the relative intensity of the band at around 1728 cm-1, since a higher intensity of that band in CAcontaining films may indicate ester formation [30, 31, 32]; and the ratio between the intensity of the band at 3300 cm-1 (I3300) and that at 1146 cm-1 (I1146), which indicates the availability of hydroxyl groups, being thus an indirect indication of the esterification reaction [31]. The band intensities have been calculated as the peak heights, measured from the baseline. The scanning electron microscopy (SEM) images of gold-coated film surfaces were taken using a Zeiss Supra 55 VP SEM (Zeiss, Oberkochen, Germany) with an acceleration voltage of 5 kV, and a magnification of 2000 x. Films were cut into 3 cm x 1 cm strips, rolled up, and inserted in a 4 mm Kel-F rotor for Nuclear Magnetic Resonance (NMR) analysis. NMR spectra were acquired on Agilent 600-MHz equipped with a 4 mm 1H –13C –15N probe at 14 kHz. The 13C cross 11
polarization under magic angle spinning (CP-MAS) was employed with an 80–100% ramp with contact time of 500 μs, high power composite pulse decoupling (Spinal 64) with 2K data points, 4 s delay between acquisitions, and 2000 scans. For contact time in CP-MAS, 80–100% ramp with contact time from 0.1 to 10 ms with 256 scans, 2K data points, and 3 s delay between acquisitions was used. The variable contact time (VCT) curves were obtained and the data was adjusted with the equation described by Kolodziejski & Klinowski [33]. The VNMRJ™ 4.0 software (Varian NMR Systems, Palo Alto, CA, USA) was used for data acquisition and processing.
3. Results and Discussion
3.1. Characterization of pectin and cellulose nanocrystals (CNCs) A high methoxyl pectin (HMP) has been extracted from banana peels, with a DM of 56% and a GAC of 73.4%. The pectin extraction resulted in a yield of 15.1 wt% on a dry AIR basis, which is within the range reported by Happi Emaga et al. [5]. The CNC yield was 7.56 wt% on a dry AIR basis. Considering an -cellulose content of 18.7 wt% for banana peels, as determined previously [6], a yield of about 40 wt% was achieved on a raw cellulose basis. The CNCs (Figure 1) presented average thickness of 4.4 nm (± 0.5 nm) and length of 240 nm (± 35 nm), resulting in an average aspect ratio of about 54 (± 3), higher than those reported for CNCs from coconut husk fiber [34, 35], cotton fiber [34], cotton linter [36], and soy hulls [37]. CNCs with aspect ratios bigger than 50 are expected to promote an especially
12
efficient reinforcing effect [38]. The theoretical percolation threshold (v), estimated according to Equation 3 [39], results in a volumetric fraction of 1.3 v%. 0.7
𝑣 = 𝐿/𝑊
(3)
Considering the densities of CNC, the extracted pectin, and glycerol as 1.6 g.cm-3 [40], 1.1 g.cm-3 (as measured by hydrostatic weighing), and 1.26 g.cm-3, respectively, the CNC theoretical weight fraction required to form a percolated network in glycerol-plasticized pectin films is about 1.8 wt%. Above this percolation threshold, CNCs are expected to form a continuous hydrogen bond-linked network, resulting in an effective improvement of mechanical properties of films [35]. The ζ, which represents the degree of dispersion of particles, was measured as −28.3 mV, within the range reported in previous studies for CNC obtained by H2SO4 hydrolysis [41, 42], thanks to the presence of negatively charged sulfate groups on the CNC surfaces [41]. Since its absolute value was higher than ~25 mV, the repulsive forces may be considered strong enough to overcome the Van der Waals attraction forces between the particles, thus the particles do not tend to aggregate [43]. 3.2. Characterization of films Figure 2 presents the film properties as functions of CNC contents and the presence or absence of CA. The tensile strength of the films (both with and without CA) was increased by addition of at least 5 wt% CNC; higher CNC contents did not improve the strength any further. The increased strength of CNC-containing films may be 13
ascribed to favorable nanocrystal–pectin interactions as well as to the reinforcing effect through stress transfer at the nanocrystal–pectin interface [44]. Similarly, the elastic modulus increased to a plateau at 7.5 wt% CNC for films without CA, and at 5 wt% CNC for CA-containing films. The elongation was significantly decreased only when the highest CNC content (10 wt%) was added. For CA-containing films, the lack of effect of CNC (at contents lower than 10 wt%) on elongation is surprising, since CNC and CA were expected to contribute to each other to impair this property; instead, the observed behaviour might be explained by the combined effects of CNC with mixed crosslinking and plasticizing effects of citric acid, previously reported in other studies [45, 46]. A recent study [47] demonstrated that CA simultaneously improved tensile strength and elongation of gelatin films, which also seems contradictory when considering CA just as a crosslinking agent. In the present study, it is possible that the simultaneous presence of CA and CNC has brought about mixed crosslinking and plasticizing effects from CA, although the presence of 10 wt% CNC seems to have reverted this in favor of the predominance of the crosslinking effect. The stabilization of tensile strength and modulus at CNC contents above 5 wt% corroborates the observations by Khan et al. [44] for CNC-reinforced chitosan films. Except for elongation, the tensile properties were not impaired by higher CNC contents, as reported in some previous studies [13, 48, 49], indicating that CNC aggregation probably did not occur or was not enough to impair overall tensile properties of the films.
14
Although the theoretical weight fraction of CNC required to form a percolated network was estimated to be only 1.8 wt%, CNC contents above 2.5 wt% have been required for an effective reinforcement, possibly because of a less than ideal CNC dispersion within the matrix [40]. The tensile strength and elastic modulus of the films obtained in this study were higher than those produced from pomegranate peel pectin and reinforced with montmorillonite [50], but inferior to those produced from commercial pectins [51]. Anyway, the tensile strength values for all films have been higher than the lower acceptable limit for packaging films (4 MPa) [52]. The formed films were almost colorless and quite translucent, except for those with 10 wt% CNC, which looked a bit opaque. Indeed, the opacity of films (Fig. 2) was only significantly increased when 10 wt% CNC were added to films (both for those with and without CA). The opacifying effect of a filler increases with its aggregation within the matrix [53, 54] and on the difference between the refractive index of the filler and the matrix [55]. The low effect of CNC on film opacity in this study may be explained by a good CNC dispersion on the pectin matrix (except for the highest CNC content), as well as by the little difference between the refractive indexes of cellulose and pectin, i.e. 1.53 [55] and 1.50 [56], respectively. The insoluble matter content of films (either with or without CA) was increased by CNC contents of 5 wt% or more. Other studies also reported that the presence of CNCs decreased film solubility [54, 57], thanks to the water insolubility of CNCs [58]. Films containing CA tended to present higher insoluble matter content than the corresponding films without CA, which suggests the occurrence of crosslinking 15
between CA and the pectin matrix. Actually, since chemical crosslinking consists of linking polymer chains by covalent bonds, forming three-dimensional networks, one of its most noticeable effects is a decreased water solubility of the crosslinked material [45, 59, 60]. Films presented decreasing water vapor permeability (WVP) with increasing CNC contents. The WVP-decreasing was effective at 2.5 wt% for films without CA, and at 5 wt% for CA-containing films, and is ascribed to the tortuous path induced by CNCs for water vapor transmission [61, 62]. The effects of CA presence are represented in Table 1 from paired t tests for each response, taking all replicates for all CNC levels into account. CA-containing films presented significantly higher strength and lower elongation, and also higher insoluble matter and lower WVP. Those findings suggest a possible crosslinking effect by CA, corroborating other studies on CA in polysaccharide films [16, 31, 45]. CA-containing films also presented impaired transparency, although citric acid has a refractive index very similar to that of pectin (around 1.50, according to information from ChemicalBook [63]), probably because citric acid presented some aggregation in the films. The FTIR spectra (Figure 3) present several bands associated with the pectin structure, such as at 3300 cm-1 for OH stretching, 1224 cm-1 for OH bending in carboxyl groups [64], 1146 cm-1 for C—O stretching in C—O—H groups [65], 1102 cm-1 and 1020 cm-1 for C—O and C—H stretching [60], and 953 cm-1 for CCH and COH bending [64]. The peaks around 1620 and 1728 cm-1 are ascribed to C=O stretching of nonesterified and methyl esterified uronic carboxyl groups, respectively 16
[66]. No specific bands were observed from CNC addition. The bands at 2940 cm-1 and 2885 cm-1, ascribed to CH stretching [63, 64, 65] suggest the presence of remaining lignin [67, 68] and/or lipids [69, 70] from pectin extraction. In a previous study from our group [6], those bands were present in banana peel powder, and their intensity decreased during the pectin extraction process, which were ascribed to partial removal of lignin and/or lipids. The intensities of the band at 1728 cm-1 was higher in CA-containing films when compared to films without CA, which may be attributed to the carbonyl band of the ester formed, suggesting crosslinking [30, 31]. Moreover, the (I3300/I1146) ratio of the CA-containing films were lower than those of corresponding films without CA, indicating a decreased availability of OH groups, which is probably related to the esterification reaction involving CA [31]. The bands at 3534 cm-1 and 3398 cm-1 of the film with 20% CA (without CNC) are related to hydroxyl stretching [71] derived from the presence of citric acid. When both CNC and CA were present (film CNC-7.5-CA-20), those bands were covered by the broad band at 3300 cm-1, probably because CNC contributed for that broad band [72]. The SEM images (Figure 4) showed that the presence of CNC did not apparently change the surface roughness of the film, except when both nanocrystals and citric acid were added, creating a visibly rougher surface, possibly induced by the formation of some complex between citric acid and cellulose [73]. The
13C
solid-state CP-MAS NMR spectral profile of films is shown in Figure 5. The
overall profiles of the spectra of films with CNC and/or CA are essentially the same as that for the control film.
13C
NMR spectra are composed mainly of pectin signals
with the C6 of galacturonic acid units ranging from 165.5 to 178.5 ppm, the C1 from 17
the anomeric portion at 93.3 to 112.0, and the remaining carbons of the pyranosidic ring at C2, C3, C4, and C5 at 65.8 to 87.1 ppm [74]. The resonances from glycerol were observed at 64.1 and 73.2 ppm [75]. The anomeric signal (93.3 to 112.0) was used as a probe for the study of the dynamic processes of the spins. This signal was deconvoluted via VNMRJ™ software, and used to fit the VCT data. Table 2 shows the contact time (TCH) and the proton spin-lattice relaxation times in the rotating frame (1H-T1ρ) obtained from the VCT experiment. The more efficient mechanism for the polarization transference (less time required) was observed for films containing both CNC and CA, resulting in stiffening of polymer chains, which explains the effects of both CNC and CA on increasing modulus and decreasing elongation of the films. In addition, the 1H-T1ρ which probes slower motions in the mid-kHz range increased with CA and CNC, which is probably associated to efficient spin diffusion within crystalline cellulose [76, 77].
4. Conclusions
Bionanocomposite films from pectin and cellulose nanocrystals (CNC) from banana peels were successfully obtained, with or without the addition of citric acid. CNC contents around 5 wt% provided the films with improved tensile properties, water resistance, and water vapor barrier. The occurrence of crosslinking between citric acid and pectin was suggested by FTIR spectra and from improved film properties resulting from the presence of citric acid in the films. Both citric acid and cellulose nanocrystals promoted stiffening of the pectin chains, as showed by NMR results.
18
Even if the water solubility of the films has been decreased by CNC and citric acid, the obtained films still presented high water solubility, and should be used for applications which do not require a high water resistance – such as for primary packaging for products which will be protected from moisture by a secondary packaging.
5. Acknowledgements
The authors gratefully acknowledge the Labex Program by Brazilian Agricultural Research Corporation (Embrapa), United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme ‘Food and Health’ (grant number BB/J004545/1), and BBSRC/EMBRAPA collaboration project (grant number BBS/E/F/00042712). Authors M.F. Rosa and E.S. Brito thank the National Counsel of Technological and Scientific Development (CNPq, Brazil) for their
Research
Productivity
Fellowships
(processes
310368/2012-0
and
302770/2015-1, respectively). We also thank Nikolaus Wellner for providing FTIR equipment and for the helpful discussions.
19
6. References
[1] BCC Research, 2013. BCC Research publishes a new report on global markets for
biodegradable
polymers.
Available
at
http://www.bccresearch.com/pressroom/pls/global-volume-biodegradable-polymersmarket-reach-3-billion-2019. [2] Yapo, B.M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of extraction conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts. Food Chemistry, 100, 1356–1364. [3]
The
World
Banana
Forum.
(2015).
Statistics
and
data.
URL
http://www.fao.org/economic/worldbananaforum/statistics/en/. Accessed 14.06.15. [4] González-Montelongo, R., Lobo, M.G., & González, M. (2010). Antioxidant activity in banana peel extracts: Testing extraction conditions and related bioactive compounds. Food Chemistry, 119, 1030-1039. [5] Happi Emaga, T., Robert, C., Ronkart, S.N., Wathelet, B., & Paquot, M. (2008). Dietary fibre components and pectin chemical features of peels during ripening in banana and plantain varieties. Bioresource Technology, 99, 4346-4354. [6] Oliveira, T.I.S., Rosa, M.F., Cavalcante, F.L., Pereira, P.H.F., Moates, G.K., Wellner, N., Mazzetto, S.E., Waldron, K.W., & Azeredo, H.M.C. (2016a). Optimization of pectin extraction from banana peels with citric acid by using response surface methodology. Food Chemistry, 198, 113—118.
20
[7] Tibolla, H., Pelissari, F.M., & Menegalli, F.C. (2014). Cellulose nanofibers produced from banana peel by chemical and enzymatic treatment. LWT – Food Science and Technology, 59, 1311-1318. [8] Pasini Cabello, S.D., Takara, E.A., Marchese, J., & Ochoa, N.A. (2015). Influence of plasticizers in pectin films: Microstructural changes. Materials Chemistry and Physics, 162, 491–497. [9] Penhasi, A., & Meidan, V.M. (2014). Preparation and characterization of in situ ionic cross-linked pectin films: Unique biodegradable polymers. Carbohydrate Polymers, 102, 254–260. [10] Silva, I.S.V., Flauzino Neto, W.P., Silvério, H.A., Pasquini, D., Andrade, M.Z., & Otaguro, H. (2015). Mechanical, thermal and barrier properties of pectin/cellulose nanocrystal nanocomposite films and their effect on the storability of strawberries (Fragaria ananassa). Polymers for Advanced Technologies, doi: 10.1002/pat.3734. [11] Jonoobi, M., Oladi, R., Davoudpour, Y., Oksman, K., Dufresne, A., Hamzeh, Y., &
Davoodi,
R.
(2015).
Different
preparation
methods
and
properties
of
nanostructured cellulose from various natural resources and residues: a review. Cellulose, 22, 935–969. [12] Xu, X., Liu, F., Jiang, L., Zhu, J.Y., Haagenson, D., & Wiesenborn, D.P. (2013). Cellulose nanocrystals vs. cellulose nanofibrils: a comparative study on their microstructures and effects as polymer reinforcing agents. ACS Applied Materials & Interfaces, 5, 2999-3009.
21
[13] Cho, M.-J., & Park, B.-D. (2011). Tensile and thermal properties of nanocellulose-reinforced poly(vinyl alcohol) nanocomposites. Journal of Industrial and Engineering Chemistry, 17, 36-40. [14] Favier, V., Canova, G.R., Cavaillé, J.Y., Chanzy, H., Dufresne, A., & Gauthier, C. (1995). Nanocomposite materials from latex and cellulose whiskers. Polymers for Advanced Technologies, 6, 351-355. [15] Bonilla, J., Talón, E., Atarés, L., Vargas, M., & Chiralt, A. (2013). Effect of the incorporation of antioxidants on physicochemical and antioxidant properties of wheat starch–chitosan films. Journal of Food Engineering, 118, 271-278. [16] Olsson, E., Hedenqvist, M.S., Johansson, C., & Järnström, L. (2013). Influence of citric acid and curing on moisture sorption, diffusion and permeability of starch films. Carbohydrate Polymers, 94, 765-772. [17] Xiaohong, X., & Yang, C.Q. (2000). FTIR spectroscopy study of the formation of cyclic anhydride intermediates of polycarboxylic acids catalyzed by sodium hypophosphite. Textile Research Journal, 70, 64-70. [18] Dastidar, T.G., & Netravali, A.N. (2012). ‘Green’ crosslinking of native starches with malonic acid and their properties. Carbohydrate Polymers, 90, 1620-628. [19] Happi Emaga, T., Ronkart, S.N., Robert, C., Wathelet, B., & Paquot, M. (2008). Characterisation of pectins extracted from banana peels (Musa AAA) under different conditions using an experimental design. Food Chemistry, 108, 463–471.
22
[20] Qiu, L.-p., Zhao, G.-l., Wu, H., Jiang, L., Li, X.-f., & Liu, J.-j. (2010). Investigation of combined effects of independent variables on extraction of pectin from banana peel using response surface methodology. Carbohydrate Polymers, 80, 326–331. [21] Pelissari, F.M., Sobral, P.J.A., & Menegalli,F.C. (2014). Isolation and characterization of cellulose nanofibers from banana peels. Cellulose, 21, 417–432. [22] Khawas, P., & Deka, S.C. (2016). Isolation and characterization of cellulose nanofibers from culinary banana peel using high-intensity ultrasonication combined with chemical treatment. Carbohydrate Polymers, 137, 608–616. [23] Waldron, K.W., & Selvendran, R.R. (1990). Composition of the cell walls of different asparagus (Asparagus officinalis) tissues. Physiologia Plantarum, 80, 568– 575. [24] Manrique, G. D., & Lajolo, F. M. (2002). FT-IR spectroscopy as a tool for measuring degree of methyl esterification in pectins isolated from ripening papaya fruit. Postharvest Biology and Technology, 25, 99–107. [25] Monsoor, M.A., Kalapathy, U., & Proctor, A. (2001). Determination of polygalacturonic acid content in pectin extracts by diffuse reflectance Fourier transform infrared spectroscopy. Food Chemistry, 74, 233-238. [26]
Cranston,
characaterization
E.D., of
&
Gray,
D.G.
polyelectrolyte
(2006).
multilayers
Morphological incorporating
and
optical
nanocrystalline
cellulose. Biomacromolecules, 7, 2522–2530.
23
[27] ASTM (2005) Standard test methods for water vapor transmission of materials. E96-05. In Annual book of ASTM standards. Philadelphia: American Society for Testing and Materials. [28] Ojagh, S.M., Rezaei, M., Razavi, S.H., & Hosseini, S.M.H. (2010). Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chemistry, 122, 161-166. [29] Irissin-Mangata, J., Bauduin, G., Boutevin, B., & Gontard, N. (2001). New plasticizers for wheat gluten films. European Polymer Journal, 37, 1533–1541. [30] Reddy, N., & Yang, Y. (2010). Citric acid cross-linking of starch films. Food Chemistry, 118, 702–711. [31] Seligra, P.G., Jaramillo, C.M., Famá, L., & Goyanes, S. (2016). Biodegradable and non-retrogradable eco-films based on starch–glycerol with citric acid as crosslinking agent. Carbohydrate Polymers, 138, 66–74. [32] 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. [33] Kolodziejski, W., & Klinowski, J. (2002). Kinetics of cross-polarization in solidstate NMR: a guide for chemists. Chemical Reviews, 102, 613-628. [34] Azeredo, H.M.C., Miranda, K.W.E., Rosa, M.F., Nascimento, D.M., & De Moura, M.R. (2012). Edible films from alginate-acerola puree reinforced with cellulose whiskers. LWT – Food Science and Technology, 46, 294–297. 24
[35] Nascimento, D.M., Almeida, J.S., Dias, A.F., Figueirêdo, M.C.B., Morais, J.P.S., Feitosa, J.P.A., & Rosa, M.F. (2014). A novel green approach for the preparation of cellulose nanowhiskers from white coir. Carbohydrate Polymers, 110, 456–463. [36] Morais, J.P.S., Rosa, M.F., Souza Filho, M.S.M., Nascimento, L.D., Nascimento, D.M., & Cassales, A.R. (2013). Extraction and characterization of nanocellulose structures from raw cotton linter. Carbohydrate Polymers, 91, 229–235. [37] Flauzino Neto, W.P., Silvério, H.A., Dantas, N.O., & Pasquini, D. (2013). Extraction and characterization of cellulose nanocrystals from agro-industrial residue – Soy hulls. Industrial Crops and Products, 42, 480–488. [38] Eichhorn, S.J., Dufresne, A., Aranguren, M., Marcovich, N.E., Capadona, J. R., Rowan, S. J., Weder, C., Thielemans, W., Roman, M., Renneckar, S., Gindl, W., Veigel, S., Keckes, J., Yano, H., Abe, K., Nogi, M., Nakagaito, A.N., Mangalam, A., Simonsen, J., Benight, A. S., Bismarck, A., Berglund, L. A., & Peijs, T. (2010). Review:
current
international
research
into
cellulose
nanofibres
and
nanocomposites. Journal of Materials Science, 45, 1–33. [39] Siqueira, G., Bras, J., & Dufresne, A. (2010). Cellulosic bionanocomposites: A review of preparation, properties and applications. Polymers, 2, 728–765. [40] Morelli, C.L., Belgacem, M.N., Branciforti, M.C., Bretas, R.E.S., Crisci, A., & Bras, J. (2016). Supramolecular aromatic interactions to enhance biodegradable film properties through incorporation of functionalized cellulose nanocrystals. Composites: Part A, 83, 80-88.
25
[41] Kargarzadeh, H., Ahmad, I., Abdullah, I., Dufresne, A., Zainudin, S.Y., & Sheltami, R.M. (2012). Effects of hydrolysis conditions on the morphology, crystallinity, and thermal stability of cellulose nanocrystals extracted from kenaf bast fibers. Cellulose, 19, 855–866. [42] Lin, N., & Dufresne, A. (2014). Surface chemistry, morphological analysis and properties of cellulose nanocrystals with gradiented sulfation degrees. Nanoscale, 6, 5384–5393. [43] Moon, J.S., Park, J.H., Lee, T.Y., Kim, Y.W., Yoo, J.B., Park, C.Y., Kim, J.M., & Jin, K.W. (2005). Transparent conductive film based on carbon nanotubes and PEDOT composites. Diamond and Related Materials, 14, 1882—1887. [44] Khan, A., Khan, R.A., Salmieri, S., Le Tien, C., Riedl, B., Bouchard, J., Chauve, G., Tan, V., Kamal, M.R., & Lacroix, M. (2012). Mechanical and barrier properties of nanocrystalline
cellulose
reinforced
chitosan
based
nanocomposite
films.
Carbohydrate Polymers, 90, 1601-1608. [45] Azeredo, H.M.C., Kontou-Vrettou, C., Moates, G.K., Wellner, N., Cross, K., Pereira, P.H.F., & Waldron, K.W. (2015). Wheat straw hemicellulose films as affected by citric acid. Food Hydrocolloids, 50, 1–6. [46] Wang, S., Ren, J., Li, W., Sun, R., & Liu, S. (2014). Properties of polyvinyl alcohol/xylan composite films with citric acid. Carbohydrate Polymers, 103, 94—99. [47] Uranga, J., Leceta, I.. Etxabide, A., Guerrero, P., & de la Caba, K. (2016). Cross-linking of fish gelatins to develop sustainable films with enhanced properties. European Polymer Journal, 78, 82–90. 26
[48] Abdollahi, M., Alboofetileh, M., Behrooz, R., Rezaei, M., & Miraki, R. (2013). Reducing water sensitivity of alginate bio-nanocomposite film using cellulose nanoparticles. International Journal of Biological Macromolecules, 54, 166-173. [49] Huq, T., Salmieri, S., Khan, A., Khan, R.A., Tien, C.L., Riedl, B., Fraschini, C., Bouchard, J., Eribe-Calderon, J., Kamal, M.R., & Lacroix, M. (2012). Nanocrystalline cellulose (NCC) reinforced alginate based biodegradable nanocomposite film. Carbohydrate Polymers, 90, 1757-1763. [50] Oliveira, T.I.S., Zea-Redondo, L., Moates, G.K., Wellner, N., Cross, K., Waldron, K.W., & Azeredo, H.M.C. (2016b). Pomegranate peel pectin films as affected by montmorillonite. Food Chemistry, 198, 107—112. [51] Lorevice, M.V., Otoni, C.G., de Moura, M.R., & Mattoso, L.H.C. (2016). Chitosan nanoparticles on the improvement of thermal, barrier, and mechanical properties of high- and low-methyl pectin films. Food Hydrocolloids, 52, 732–740. [52] Tajeddin, B., Rahman, R.A., & Abdulah L.C. (2010). The effect of polyethylene glycol
on
the
characteristics
of
kenaf
cellulose/low-density
polyethylene
biocomposites. International Journal of Biological Macromolecules, 47, 292—297. [53] Castillo, L., López, O., López, C., Zaritzky, N., García, M.A., Barbosa, S., & Villar, M. (2013). Thermoplastic starch films reinforced with talc nanoparticles. Carbohydrate Polymers, 95, 664—674. [54] Pereda, M., Dufresne, A., Aranguren, M.I., & Marcovich, N.E. (2014). Polyelectrolyte films based on chitosan/olive oil and reinforced with cellulose nanocrystals. Carbohydrate Polymers, 101, 1018–1026. 27
[55] Bardet, R., Belgacem, M.N., & Bras, J. (2013). Different strategies for obtaining high opacity films of MFC with TiO2 pigments. Cellulose, 20, 3025–3037. [56] Donaldson, L.A. (1991). Seasonal changes in lignin distribution during tracheid development in Pinus radiata D. Don. Wood Science and Technology, 25¸15–24. [57] Slavutsky, A.M., & Bertuzzi, M.A. (2014). Water barrier properties of starch films reinforced
with
cellulose
nanocrystals
obtained
from
sugarcane
bagasse.
Carbohydrate Polymers, 110, 53—61. [58] George, J. & Sabapathi, S.N. (2015). Cellulose nanocrystals: synthesis, functional properties, and applications. Nanotechnology, Science and Applications, 8, 45—54. [59] Balaguer, M.P., Gómez-Estaca, J., Gavara, R., & Hernandez-Munoz, P. (2011). Functional properties of bioplastics made from wheat gliadins modified with cinnamaldehyde. Journal of Agricultural and Food Chemistry, 59, 6689–6695. [60] Menzel, C., Olsson, E., Plivelic, T.S., Andersson, R., Johansson, C., Kuktaite, R., Järnström, L., & Koch, K. (2013). Molecular structure of citric acid cross-linked starch films. Carbohydrate Polymers, 96, 270—276. [61] El Miri, N., Abdelouahdi, K., Barakat, A., Zahouily, M., Fihri, A., Solhy, A., & El Achaby, M. (2015). Bio-nanocomposite films reinforced with cellulose nanocrystals: Rheology of film-forming solutions, transparency, water vapor barrier and tensile properties of films. Carbohydrate Polymers, 129, 156–167.
28
[62] Oun, A.A., & Rhim, J.-W. (2015). Preparation and characterization of sodium carboxymethyl
cellulose/cotton
linter
cellulose
nanofibril
composite
films.
Carbohydrate Polymers, 127, 101–109. [63]
ChemicalBook,
CAS
DataBase
List.
Citric
acid.
Available
at
http://www.chemicalbook.com/ChemicalProductProperty_EN_CB9854361.htm, assessed on May 11th, 2016). [64] Synytsya, A., Čopíková, J., Matĕjka, P., & Machovič, V. (2003). Fourier transform Raman and infrared spectroscopy of pectins. Carbohydrate Polymers, 54, 97-106. [65] Yu, J.G., Wang, N., & Ma, X.F. (2005). The effects of citric acid on the properties of thermoplastic starch plasticized by glycerol. Starch/Stärke, 57, 494–504. [66] Peng, X., Mu, T., Zhang, M., Sun, H., Chen, J., & Yu, M. (2016). Effects of pH and high hydrostatic pressure on the structural and rheological properties of sugar beet pectin. Food Hydrocolloids, 60, 161—169. [67] Dawy, M., Shabaka, A.A., & Nada, A.M.A. (1998). Molecular structure and dielectric properties of some treated lignins. Polymer Degradation and Stability, 62, 455-462. [68] Diehl, B.G., Brown, N.R., Frantz, C.W., Lumadue, M.R., & Cannon, F. (2013). Effects of pyrolysis temperature on the chemical composition of refined softwood and hardwood lignins. Carbon, 60, 531–537.
29
[69] Marcott, C., Lo, M., Kjoller, K., Fiat, F., Baghdadli, N., Balooch, G., & Luengo, G.S. (2014). Localization of human hair structural lipids using nanoscale infrared spectroscopy and imaging. Applied Spectroscopy, 68, 564–569. [70] Derenne, A., Claessens, T., Conus, C., & Goormaghtigh, E. (2013). Infrared spectroscopy of membrane lipids. In G. C. K. Roberts (Ed.), Encyclopedia of biophysics (1074—1081). Berlin: Springer. [71] Kloprogge, J.T., Hickey, L., & Frost, R.L. (2004). FT-Raman and FT-IR spectroscopic study of synthetic Mg/Zn/Al-hydrotalcites. Journal of Raman Spectroscopy, 35, 967—974. [72] Le Tien, C., Letendre, M., Ispas-Szabo, P., Mateescu, M.A., Delmas-Patterson, G., Yu, H.L., & Lacroix, M. (2000). [73] Edwards, J.V., Eggleston, G., Yager, D.R., Cohen, I.K., Diegelmann, R.F., & Bopp,
A.F.
(2002).
Design,
preparation
and
assessment
of
citrate-linked
monosaccharide cellulose conjugates with elastase-lowering activity. Carbohydrate Polymers, 50, 305–314. [74] Zhu, X., Liu, B., Zheng, S., & Gao, Y. (2014). Quantitative and structure analysis of pectin in tobacco by
13C
CP/MAS NMR spectroscopy. Analytical Methods, 6,
6407-6413. [75] Wishart, D.S., Tzur, D., Knox, C., Eisner, R., Guo, A.C., Young, N., Cheng, D., Jewell, K., Arndt, D.,Sawhney, S., Fung, C., Nikolai, L., Lewis, M., Coutouly, M.-A., Forsythe, I., Tang, P., Shrivastava, S., Jeroncic, K., Stothard, P., Amegbey, G., Block, D., Hau, D.D., Wagner, J.,
Miniaci, J., Clements, M., Gebremedhin, M., Guo, 30
N., Zhang, Y., Duggan, G.E., MacInnis, G.D., Weljie, A.M., Dowlatabadi, R., Bamforth, F., Clive, D., Greiner, R., Li, L., Marrie, T., Sykes, B.D., Vogel, H.J., & Querengesser, L. (2007). HMDB: the Human metabolome database. Nucleic Acids Research, 35, D521-D526. [76] Tang, H.-R., Wang, Y.-L., & Belton, P.S. (2000). 13C CPMAS studies of plant cell wall materials and model systems using proton relaxation-induced spectral editing techniques. Solid State Nuclear Magnetic Resonance, 15, 239–248. [77] Okushita, K., Komatsu, T., Chikayama, E., & Kikuchi, J. (2012). Statistical approach for solid-state NMR spectra of cellulose derived from a series of variable parameters. Polymer Journal, 44, 895–900.
31
Figure captions: Figure 1. Transmission electron micrograph (TEM) of celulose nanocrystals from banana peels. Figure 2. Film properties as functions of CNC contents and presence/absence of citric acid. Values in the same line (color) followed by the same letter are not significantly different (Tukey, p< 0.05). Values in parentheses are standard deviations. Figure 3. FTIR spectra of pectin films with different cellulose nanocrystals (CNC) and citric acid (CA) contents, as well as of pure pectin and CNC suspension. Films identified as CNC-X-CA-Y, X and Y indicating the cellulose nanocrystal and citric acid contents (%). Figure 4. Scanning electron micrographs from (A) pectin control film; (B) pectin film with 7.5 wt% cellulose nanocrystals; (C) pectin film with 20 wt% citric acid; (D) pectin film with 20 wt% citric acid and 7.5 wt% cellulose nanocrystals. Figure 5.
13C
CP-MAS spectra of films (identified as CNC-X-CA-Y, X and Y
indicating the percent cellulose nanocrystal and citric acid contents, respectively).
32
Fig 1
Fig 2 33
Fig 3
Fig 4
34
Fig 5
35
Table 1. Differences between films without and with citric acid, according to paired t tests for all replicates at all CNC levels.
Averages* Response
t
p
7.92 1.21
-2.88
0.010
4.69 0.84
4.26 0.77
2.40
0.030
Elastic modulus (MPa)
1586 487
1714 452
-1.67
0.115
Opacity (A.nm.mm-1)
2246.3 238.6
2412.0 248.6
-3.56
< 0.01
Insoluble matter (%)
13.13 2.87
14.75 3.91
-3.24
< 0.01
WVP (g.mm.kPa-1.h-1.m-2)
3.31 0.36
3.10 0.33
3.10
< 0.01
Without citric acid
With citric acid
Tensile strength (MPa)
7.36 1.15
Elongation at break (%)
*Averages ( standard deviation) of at least 25 values (5 CNC contents, at least 5 replicates).
Table 2. Contact time (TCH) and proton spin-lattice relaxation times in the rotating frame (1H-T1ρ) of some films.
Film*
TCH (ms)
T1ρ (s)
CNC-0-CA-0
58.6
6.8
CNC-10-CA-0
63.2
7.6
CNC-0-CA-20
85.78
7.8
CNC-10-CA-20
44.47
13.15
* Films identified as CNC-X-CA-Y, X and Y indicating the cellulose nanocrystal and citric acid contents (%).
36
S0141-8130(16)31273-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.068 BIOMAC 7234
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
17-8-2016 10-3-2017 12-3-2017
Please cite this article as: T´ulio ´Italo S.Oliveira, Morsyleide F.Rosa, Michael J.Ridout, Kathryn Cross, Edy S.Brito, Lorena M.A.Silva, Selma E.Mazzetto, Keith W.Waldron, Henriette M.C.Azeredo, Bionanocomposite films based on polysaccharides from banana peels, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.068 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bionanocomposite films based on polysaccharides from banana peels
Túlio Ítalo S. Oliveiraa, Morsyleide F. Rosab, Michael J. Ridoutc, Kathryn Crossc, Edy S. Britob, Lorena M. A. Silvab, Selma E. Mazzettoa, Keith W. Waldronc, Henriette M. C. Azeredob,*
aFederal
University of Ceara, Organic and Inorganic Chemistry Department, Campus
Pici, 60440-900, Fortaleza, CE, Brazil; bEmbrapa Tropical Agroindustry, R. Dra. Sara Mesquita, 2270, Pici, 60511-110, Fortaleza, CE, Brazil; cInstitute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK. *Corresponding author, [email protected].
1
Abstract
Pectin and cellulose nanocrystals (CNCs) isolated from banana peels were used to prepare films. The effects of a reinforcing phase (CNCs) and a crosslinker (citric acid, CA) on properties of pectin films were studied. Glycerol-plasticized films were prepared by casting, with different CNC contents (0–10 wt%), with or without CA. Overall tensile properties were improved by intermediate CNC contents (around 5 wt%). The water resistance and water vapor barrier properties were also enhanced by CNC. Evidences were found from Fourier Transform Infrared (FTIR) spectra supporting the occurrence of crosslinking by CA. Additionally, the tensile strength, water resistance and barrier to water vapor were improved by the presence of CA. The
13C
ssNMR spectra indicated that both CA and CNC promoted stiffening of the
polymer chains. Keywords: polysaccharides; biopolymers; bionanocomposites; food packaging.
2
1. Introduction
Concerns about environmental impacts from food packaging waste have motivated development of biodegradable materials, mostly from renewable sources. The global market for biodegradable polymers has increased from 0.4 to 1.3 billion pounds between 2006 and 2013 [1]. Pectins, present in plant primary cell walls and middle lamellae, have a main backbone consisting of -1,4-linked galacturonic acids with variable degree of methoxylation (DM) of their carboxylic acid residues. According to their DM, pectins are classified as high methoxyl pectins (HMP) or low methoxyl pectins (LMP), having DM 50 and DM < 50, respectively [2]. The global annual production of bananas (Musa acuminata, AAA Group) surpassed 100 million tons in 2011, Brazil being the fourth producer [3]. Banana peels (30% of fruit weight) are usually discarded, causing pollution because of their susceptibility to microbial degradation [4]. They have been presented as sources of pectin [5, 6] as well as cellulose [6, 7], which makes their proper exploitation interesting from the perspective of valorization of by-products for non-food applications in a biorefinery context, transforming them into value-added products, decreasing their negative environmental impacts, and enhancing their economic feasibility. Pectins from other sources have been used for development of biodegradable films [8, 9]. However, the use of polysaccharide films for food packaging is still limited due to their poor tensile and barrier properties when compared to those of petroleumderived polymers. The incorporation of nanoreinforcements such as cellulose nanocrystals (CNCs) has been suggested to improve the performance of pectin films 3
[10]. CNCs are needlelike crystals measuring 4-25 nm in diameter and 100-1,000 nm in length [11], often isolated by processes including acid hydrolysis [12]. The most studied effects of CNCs on polymer matrices are on tensile properties, due to the high strength and stiffness of CNCs [13] and to mechanical percolation yielded by CNC hydrogen bonding interactions [14]. Another limitation of polysaccharide-based films is their strong hydrophilic character, which makes them dissolve in contact with water, limiting their applications. Citric acid has been reported to crosslink polysaccharide films by forming covalent di-ester linkages between two of their carboxyl groups and hydroxyl groups of different polysaccharide chains [15, 16]. Carboxyl groups of citric acid form cyclic anhydrides, which react further with hydroxyls from the polysaccharide [17]. The reaction is favored by curing the dried films, typically above 100˚C [16, 18]. Although banana peels have been reported in previous studies as a source of pectins [5, 6, 19, 20] and cellulose nanostructures [7, 21, 22], to our knowledge, this is the first study to propose the development of films from pectin and cellulose nanocrystals isolated from banana peels. Considering that both nanoreinforcing and crosslinking agents have been reported to improve film properties by different mechanisms, this study was conducted to evaluate the combined effects of a nanoreinforcement (CNC) and a crosslinker (citric acid) on tensile, barrier, and water resistance properties of films from banana peel pectin, in order to evaluate if that combination of additives results in films with improved properties when compared with the individual use of each additive.
4
2. Materials and Methods
2.1. Extraction and characterization of pectin from banana peels Banana peels at ripening stage 6 (fully ripe bananas) were collected from Cavendish bananas (Musa AAA group, Cavendish subgroup) purchased in the local market (Norwich, UK). The peels were immersed in Na2S2O5 solution (1% w/v) for 24 h, oven-dried at 60°C for 24 h, and milled to 0.5 mm in a Retsch Brinkmann ZM-1 centrifugal grinding mill (Retsch GmbH, Haan, Germany). The alcohol insoluble residue (AIR) preparation was based on the method by Waldron & Selvendran [23], with some modifications. 100 g of milled peels were washed three times with ethanol to remove alcohol soluble phenolics (firstly with 600 mL boiling 70 v% ethanol for 5 min, then with 600 mL boiling absolute ethanol for 5 min, the third time with 600 mL absolute ethanol for 5 min), then washed in 200 mL acetone. Between washings, the material was filtered through a 10 µm nylon mesh. The AIR was air dried at room temperature, and used thereafter for extraction of both pectin and cellulose nanocrystals. The pectin was extracted with a citric acid solution at pH 2.0 (AIR: solution ratio, 1:20, w/v) at 87°C for 160 min under stirring (150 rpm), and centrifuged at 1,850 g for 30 min at 10°C in a Beckman JS-4.2 centrifuge (Beckman Coulter, Brea, USA). The supernatant was vacuum filtered, had its pH adjusted to 3.5 (minimum pectin solubility) with KOH, and was added with absolute ethanol (1:1, v/v). The suspension was stirred for 30 min and left to precipitate at 4°C for 2 h. After centrifugation (15 min, 4°C, 2,520 g, in the same Beckman JS-4.2 centrifuge), the pellet was collected, 5
washed with ethanol 70 v%, centrifuged again (20 min, 4°C, 2,520 g), and dried at room temperature. It was then stirred in distilled water with a Ystral X10/25 homogenizer (Ballrechtar-Dottingen, Germany) for 20 min, had its pH adjusted to 7, and was again dried and milled by using a basic mill (A10, IKA GmbH, Germany). The DM and the galacturonic acid content (GAC) of the pectin were determined from Fourier Transform Infrared (FTIR) spectra collected in the frequency range of 4000– 800 cm-1 (128 scans at 2 cm−1 resolution) on a Nicolet Magna-IR 860 FTIR spectrometer (Thermo Nicolet, Madison, WI, USA). Samples were placed on a GoldenGate diamond ATR accessory (Specac, Orpington, Kent), the empty crystal used as reference. The DM was determined as described elsewhere [24], with some modifications. FTIR spectra were firstly obtained from pectin standards with known DM values (31%, 67%, and 89%) from Sigma (Steinheim, Germany) and four additional standards obtained by blending the initial standards (for DM values of 40, 49, 60, and 78). The DM determination was based on the band areas at 1700-1750 cm-1 (methyl esterified uronic acids, EUA) and 1600-1630 cm-1 (free uronic acids, FUA), calculated by using multiple peak fit function/Gaussian fitting on Origin Pro 9 (OriginLab, Northampton, USA). The calibration curve (Equation 1), obtained by plotting DM of the standards against the ratio between EUA peak area (AEUA) over the sum of EUA and FUA peak areas (AEUA + AFUA), was used to determine the DM of banana peel pectin.
DM (%) 126.3
AEUA 2.493 AEUA AFUA
( R 2 0.974)
(1)
6
The GAC determination was modified from a previously described method [25]. A set of
10 calibration galacturonic acid standards was prepared by blending
polygalacturonic acid (95%, 81325, Fluka) with cellulose (Sigmacell type 20, Sigma) to several final GAC values (0 to 90 wt%). Peak areas were measured as the total carbonyl peak area above the baseline between 1840 cm -1 and 1550 cm-1 (A15501840).
The calibration curve (Equation 2), obtained by plotting GAC against A1550-1840,
was used to determine GAC of banana peel pectin. GAC (%) 0.7934 A15501840 - 0.1985
( R 2 0.951)
(2)
2.2. Extraction and characterization of cellulose nanocrystals (CNC) Acetosolv pulping was conducted by suspending AIR in a mixture of 93 wt% acetic acid and 0.3 wt% HCl in distilled water, in a 1:10 ratio (w/v, AIR:solution), in a flat bottom flask with reflux at 115 °C under constant stirring (150 rpm) for 3 h. The mixture was filtered through a 28 μm filter paper, resulting in a lignin-rich black liquor and the acetosolv pulp (partly delignified fibers). The acetosolv pulp was rinsed with hot (80°C) acetic acid (99.7%) until the washed liquid was colorless, then washed with distilled water until pH 6.5, and finally oven-dried at 45°C until constant weight. The acetosolv pulp was bleached in two steps. The first one was conducted in an aqueous solution containing H2O2 6% (v/v) and NaOH 4% (w/v) in a beaker at 70°C in a ratio of 1:90 (pulp:solution, w/v) with constant stirring (150 rpm) for 3 h. Then, after being washed with distilled water to pH 7 and oven dried at 60°C for 24h, the pulp was immersed in KOH 6% (w/v) in a 1:20 ratio (w/v, pulp:solution) at 80°C with constant stirring (150 rpm) for 90 min. 7
The acid hydrolysis was then conducted as described previously [26], with some modifications. The bleached acetosolv pulp was stirred (200 rpm) in a 30 v% H2SO4 solution at 45°C, in a 1:20 ratio (w/v, pulp: solution) for 150 min. Immediately following hydrolysis, the suspension was diluted 10-fold with deionized water to quench the reaction, then submitted to three cycles including centrifugation (9,800 g for 15 min at 20°C, in an Avanti J-25 centrifuge, Beckman Coulter, Brea, USA), dilution 10-fold with deionized water, and sonication for 2 min in a probe Branson sonicator (Digital Sonifier 250, Branson Ultrasonics, Danbury, USA) at 20 kHz. After those centrifugation-dilution-sonication cycles, the CNC suspension was dialysed against deionized water for 48 h with four changes of water. The final concentration of the CNC suspension was 0.22 g/100 mL. The zeta-potential (ζ) of CNCs was measured in triplicate using a Zetasizer Nano ZS 3000 (Malvern Instruments Ltd., Worcestershire, UK) for CNC suspensions diluted to 0.1 wt% with deionized water. For the transmission electron microscopy (TEM) images, previously sonicated CNC suspensions were placed onto Formvar/carbon coated grids and negatively stained with 2% uranyl acetate. The samples were imaged using a Tecnai 20 Transmission Electron Microscope (FEI, Hillsboro, USA), with an acceleration voltage of 200 kV, and a magnification of 57500 x. The CNC dimensions (length and diameter) were taken as averages of 100 measurements taken by using the scale bar embedded in the image. 2.3. Film preparation
8
Ten films were prepared, with different CNC contents (0, 2.5, 5, 7.5, and 10 wt% on a dry bionanocomposite basis), with or without citric acid (20 wt% on a dry bionanocomposite basis, as defined from preliminary tests), hereafter referred to as CNC-X-CA-Y, X and Y indicating the percent CNC and citric acid (CA) contents, respectively. For each film, the CNC dispersion (in a volume determined according to its final intended concentration) was collected and added with 4.5 g pectin, 1.35 g glycerol (added as a plasticizer), citric acid, and distilled water, whose volume was calculated to provide a solid content of 2.5 g/100 mL. The film forming dispersion was homogenized in an X10/25 homogenizer (Ystral, Ballrechtar-Dottingen, Germany) for 30 min. Air bubbles were removed under vacuum, and the films were cast on petri dishes to a dry thickness of 0.10 mm, and left to dry at 40˚C in a fan oven (Memmert, Schwabach, Germany) for 16 h. The films added with CA were then cured at 150˚C for 10 minutes using the same fan oven. The dried films were detached from the petri dishes and conditioned (50% RH, 24˚C) in an environmental chamber (Weiss Gallenkamp, Loughborough, UK) for 40 h before the analyses. 2.4. Characterization of films The water vapor permeability (WVP) determination was modified from the ASTM method E96-05 [27] for five samples. The test films had their thicknesses measured (micrometer screw gauge, Moore & Wright, Sheffield, UK) to the nearest 0.01 mm at six random locations, and were sealed as patches onto acrylic permeation cells (2.4 cm in diameter and 1 cm in height) containing 2 mL distilled water. The cells were placed in a desiccator connected to tubes providing a steady flow of dried air (less 9
than 1% RH) from a Balston 75-60 air drier at 24 °C, and were weighed eight times over a 24-h period. For each sample, measurements were taken in at least five replicates. The insoluble matter was determined in quadruplicate on 2 cm x 2 cm film pieces, according to a previously proposed method [28], with modifications. Previously dried and weighed samples were immersed in 50 mL distilled water for 6 h at 25˚C under stirring (150 rpm). The dry weight of the remaining film pieces was obtained after filtration on previously dried and weighed filter paper, and was used to calculate insoluble matter as a percentage of the initial dry weight. The dry weights were determined after drying at 103˚C for 24 h using a fan oven (Memmert, Schwabach, Germany). Tensile tests were conducted (with at least five replicates) on 8 mm x 50 mm specimens on a Texture Analyzer TA.XT Plus (Stable Micro Systems, Godalming, UK) equipped with A/TG Tensile Grips and a 5 kg load cell. The sample thicknesses were determined with a micrometer to the nearest 0.01 mm at 6 random locations. The initial grip separation and crosshead speed were set to 40 mm and 1 mm/s, respectively. Film opacity was determined (in quadruplicate) as described elsewhere [29]. Film strips (1 cm x 5 cm) were placed on the internal side of a Varian Cary 50 UV–vis spectrophotometer
cell
(Agilent
Technologies,
Santa
Clara,
CA,
USA),
perpendicularly to the light beam. The absorbance spectrum (400–800 nm) of film samples was recorded, and opacity was defined as the area under the recorded
10
curve (estimated by the linear trapezoidal rule) and expressed as absorbance units x nanometers (wavelength) per millimeter (film thickness) (A.nm.mm -1). The significance of differences between films without and with CA for tensile properties, insoluble matter, and WVP was established by paired t tests for each response, considering all replicates for all CNC levels. Fourier Transform infrared (FTIR) spectra (128 scans at 2 cm −1 resolution for a spectral range from 4000-800 cm−1) were recorded using a Nicolet Magna-IR 860 FTIR spectrometer (Thermo Nicolet, Madison, WI, USA). Samples were placed on a GoldenGate diamond ATR accessory (Specac, Orpington, Kent). The empty crystal was used as reference. The crosslinking occurrence was assessed by two comparisons between corresponding films with and without CA: the relative intensity of the band at around 1728 cm-1, since a higher intensity of that band in CAcontaining films may indicate ester formation [30, 31, 32]; and the ratio between the intensity of the band at 3300 cm-1 (I3300) and that at 1146 cm-1 (I1146), which indicates the availability of hydroxyl groups, being thus an indirect indication of the esterification reaction [31]. The band intensities have been calculated as the peak heights, measured from the baseline. The scanning electron microscopy (SEM) images of gold-coated film surfaces were taken using a Zeiss Supra 55 VP SEM (Zeiss, Oberkochen, Germany) with an acceleration voltage of 5 kV, and a magnification of 2000 x. Films were cut into 3 cm x 1 cm strips, rolled up, and inserted in a 4 mm Kel-F rotor for Nuclear Magnetic Resonance (NMR) analysis. NMR spectra were acquired on Agilent 600-MHz equipped with a 4 mm 1H –13C –15N probe at 14 kHz. The 13C cross 11
polarization under magic angle spinning (CP-MAS) was employed with an 80–100% ramp with contact time of 500 μs, high power composite pulse decoupling (Spinal 64) with 2K data points, 4 s delay between acquisitions, and 2000 scans. For contact time in CP-MAS, 80–100% ramp with contact time from 0.1 to 10 ms with 256 scans, 2K data points, and 3 s delay between acquisitions was used. The variable contact time (VCT) curves were obtained and the data was adjusted with the equation described by Kolodziejski & Klinowski [33]. The VNMRJ™ 4.0 software (Varian NMR Systems, Palo Alto, CA, USA) was used for data acquisition and processing.
3. Results and Discussion
3.1. Characterization of pectin and cellulose nanocrystals (CNCs) A high methoxyl pectin (HMP) has been extracted from banana peels, with a DM of 56% and a GAC of 73.4%. The pectin extraction resulted in a yield of 15.1 wt% on a dry AIR basis, which is within the range reported by Happi Emaga et al. [5]. The CNC yield was 7.56 wt% on a dry AIR basis. Considering an -cellulose content of 18.7 wt% for banana peels, as determined previously [6], a yield of about 40 wt% was achieved on a raw cellulose basis. The CNCs (Figure 1) presented average thickness of 4.4 nm (± 0.5 nm) and length of 240 nm (± 35 nm), resulting in an average aspect ratio of about 54 (± 3), higher than those reported for CNCs from coconut husk fiber [34, 35], cotton fiber [34], cotton linter [36], and soy hulls [37]. CNCs with aspect ratios bigger than 50 are expected to promote an especially
12
efficient reinforcing effect [38]. The theoretical percolation threshold (v), estimated according to Equation 3 [39], results in a volumetric fraction of 1.3 v%. 0.7
𝑣 = 𝐿/𝑊
(3)
Considering the densities of CNC, the extracted pectin, and glycerol as 1.6 g.cm-3 [40], 1.1 g.cm-3 (as measured by hydrostatic weighing), and 1.26 g.cm-3, respectively, the CNC theoretical weight fraction required to form a percolated network in glycerol-plasticized pectin films is about 1.8 wt%. Above this percolation threshold, CNCs are expected to form a continuous hydrogen bond-linked network, resulting in an effective improvement of mechanical properties of films [35]. The ζ, which represents the degree of dispersion of particles, was measured as −28.3 mV, within the range reported in previous studies for CNC obtained by H2SO4 hydrolysis [41, 42], thanks to the presence of negatively charged sulfate groups on the CNC surfaces [41]. Since its absolute value was higher than ~25 mV, the repulsive forces may be considered strong enough to overcome the Van der Waals attraction forces between the particles, thus the particles do not tend to aggregate [43]. 3.2. Characterization of films Figure 2 presents the film properties as functions of CNC contents and the presence or absence of CA. The tensile strength of the films (both with and without CA) was increased by addition of at least 5 wt% CNC; higher CNC contents did not improve the strength any further. The increased strength of CNC-containing films may be 13
ascribed to favorable nanocrystal–pectin interactions as well as to the reinforcing effect through stress transfer at the nanocrystal–pectin interface [44]. Similarly, the elastic modulus increased to a plateau at 7.5 wt% CNC for films without CA, and at 5 wt% CNC for CA-containing films. The elongation was significantly decreased only when the highest CNC content (10 wt%) was added. For CA-containing films, the lack of effect of CNC (at contents lower than 10 wt%) on elongation is surprising, since CNC and CA were expected to contribute to each other to impair this property; instead, the observed behaviour might be explained by the combined effects of CNC with mixed crosslinking and plasticizing effects of citric acid, previously reported in other studies [45, 46]. A recent study [47] demonstrated that CA simultaneously improved tensile strength and elongation of gelatin films, which also seems contradictory when considering CA just as a crosslinking agent. In the present study, it is possible that the simultaneous presence of CA and CNC has brought about mixed crosslinking and plasticizing effects from CA, although the presence of 10 wt% CNC seems to have reverted this in favor of the predominance of the crosslinking effect. The stabilization of tensile strength and modulus at CNC contents above 5 wt% corroborates the observations by Khan et al. [44] for CNC-reinforced chitosan films. Except for elongation, the tensile properties were not impaired by higher CNC contents, as reported in some previous studies [13, 48, 49], indicating that CNC aggregation probably did not occur or was not enough to impair overall tensile properties of the films.
14
Although the theoretical weight fraction of CNC required to form a percolated network was estimated to be only 1.8 wt%, CNC contents above 2.5 wt% have been required for an effective reinforcement, possibly because of a less than ideal CNC dispersion within the matrix [40]. The tensile strength and elastic modulus of the films obtained in this study were higher than those produced from pomegranate peel pectin and reinforced with montmorillonite [50], but inferior to those produced from commercial pectins [51]. Anyway, the tensile strength values for all films have been higher than the lower acceptable limit for packaging films (4 MPa) [52]. The formed films were almost colorless and quite translucent, except for those with 10 wt% CNC, which looked a bit opaque. Indeed, the opacity of films (Fig. 2) was only significantly increased when 10 wt% CNC were added to films (both for those with and without CA). The opacifying effect of a filler increases with its aggregation within the matrix [53, 54] and on the difference between the refractive index of the filler and the matrix [55]. The low effect of CNC on film opacity in this study may be explained by a good CNC dispersion on the pectin matrix (except for the highest CNC content), as well as by the little difference between the refractive indexes of cellulose and pectin, i.e. 1.53 [55] and 1.50 [56], respectively. The insoluble matter content of films (either with or without CA) was increased by CNC contents of 5 wt% or more. Other studies also reported that the presence of CNCs decreased film solubility [54, 57], thanks to the water insolubility of CNCs [58]. Films containing CA tended to present higher insoluble matter content than the corresponding films without CA, which suggests the occurrence of crosslinking 15
between CA and the pectin matrix. Actually, since chemical crosslinking consists of linking polymer chains by covalent bonds, forming three-dimensional networks, one of its most noticeable effects is a decreased water solubility of the crosslinked material [45, 59, 60]. Films presented decreasing water vapor permeability (WVP) with increasing CNC contents. The WVP-decreasing was effective at 2.5 wt% for films without CA, and at 5 wt% for CA-containing films, and is ascribed to the tortuous path induced by CNCs for water vapor transmission [61, 62]. The effects of CA presence are represented in Table 1 from paired t tests for each response, taking all replicates for all CNC levels into account. CA-containing films presented significantly higher strength and lower elongation, and also higher insoluble matter and lower WVP. Those findings suggest a possible crosslinking effect by CA, corroborating other studies on CA in polysaccharide films [16, 31, 45]. CA-containing films also presented impaired transparency, although citric acid has a refractive index very similar to that of pectin (around 1.50, according to information from ChemicalBook [63]), probably because citric acid presented some aggregation in the films. The FTIR spectra (Figure 3) present several bands associated with the pectin structure, such as at 3300 cm-1 for OH stretching, 1224 cm-1 for OH bending in carboxyl groups [64], 1146 cm-1 for C—O stretching in C—O—H groups [65], 1102 cm-1 and 1020 cm-1 for C—O and C—H stretching [60], and 953 cm-1 for CCH and COH bending [64]. The peaks around 1620 and 1728 cm-1 are ascribed to C=O stretching of nonesterified and methyl esterified uronic carboxyl groups, respectively 16
[66]. No specific bands were observed from CNC addition. The bands at 2940 cm-1 and 2885 cm-1, ascribed to CH stretching [63, 64, 65] suggest the presence of remaining lignin [67, 68] and/or lipids [69, 70] from pectin extraction. In a previous study from our group [6], those bands were present in banana peel powder, and their intensity decreased during the pectin extraction process, which were ascribed to partial removal of lignin and/or lipids. The intensities of the band at 1728 cm-1 was higher in CA-containing films when compared to films without CA, which may be attributed to the carbonyl band of the ester formed, suggesting crosslinking [30, 31]. Moreover, the (I3300/I1146) ratio of the CA-containing films were lower than those of corresponding films without CA, indicating a decreased availability of OH groups, which is probably related to the esterification reaction involving CA [31]. The bands at 3534 cm-1 and 3398 cm-1 of the film with 20% CA (without CNC) are related to hydroxyl stretching [71] derived from the presence of citric acid. When both CNC and CA were present (film CNC-7.5-CA-20), those bands were covered by the broad band at 3300 cm-1, probably because CNC contributed for that broad band [72]. The SEM images (Figure 4) showed that the presence of CNC did not apparently change the surface roughness of the film, except when both nanocrystals and citric acid were added, creating a visibly rougher surface, possibly induced by the formation of some complex between citric acid and cellulose [73]. The
13C
solid-state CP-MAS NMR spectral profile of films is shown in Figure 5. The
overall profiles of the spectra of films with CNC and/or CA are essentially the same as that for the control film.
13C
NMR spectra are composed mainly of pectin signals
with the C6 of galacturonic acid units ranging from 165.5 to 178.5 ppm, the C1 from 17
the anomeric portion at 93.3 to 112.0, and the remaining carbons of the pyranosidic ring at C2, C3, C4, and C5 at 65.8 to 87.1 ppm [74]. The resonances from glycerol were observed at 64.1 and 73.2 ppm [75]. The anomeric signal (93.3 to 112.0) was used as a probe for the study of the dynamic processes of the spins. This signal was deconvoluted via VNMRJ™ software, and used to fit the VCT data. Table 2 shows the contact time (TCH) and the proton spin-lattice relaxation times in the rotating frame (1H-T1ρ) obtained from the VCT experiment. The more efficient mechanism for the polarization transference (less time required) was observed for films containing both CNC and CA, resulting in stiffening of polymer chains, which explains the effects of both CNC and CA on increasing modulus and decreasing elongation of the films. In addition, the 1H-T1ρ which probes slower motions in the mid-kHz range increased with CA and CNC, which is probably associated to efficient spin diffusion within crystalline cellulose [76, 77].
4. Conclusions
Bionanocomposite films from pectin and cellulose nanocrystals (CNC) from banana peels were successfully obtained, with or without the addition of citric acid. CNC contents around 5 wt% provided the films with improved tensile properties, water resistance, and water vapor barrier. The occurrence of crosslinking between citric acid and pectin was suggested by FTIR spectra and from improved film properties resulting from the presence of citric acid in the films. Both citric acid and cellulose nanocrystals promoted stiffening of the pectin chains, as showed by NMR results.
18
Even if the water solubility of the films has been decreased by CNC and citric acid, the obtained films still presented high water solubility, and should be used for applications which do not require a high water resistance – such as for primary packaging for products which will be protected from moisture by a secondary packaging.
5. Acknowledgements
The authors gratefully acknowledge the Labex Program by Brazilian Agricultural Research Corporation (Embrapa), United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme ‘Food and Health’ (grant number BB/J004545/1), and BBSRC/EMBRAPA collaboration project (grant number BBS/E/F/00042712). Authors M.F. Rosa and E.S. Brito thank the National Counsel of Technological and Scientific Development (CNPq, Brazil) for their
Research
Productivity
Fellowships
(processes
310368/2012-0
and
302770/2015-1, respectively). We also thank Nikolaus Wellner for providing FTIR equipment and for the helpful discussions.
19
6. References
[1] BCC Research, 2013. BCC Research publishes a new report on global markets for
biodegradable
polymers.
Available
at
http://www.bccresearch.com/pressroom/pls/global-volume-biodegradable-polymersmarket-reach-3-billion-2019. [2] Yapo, B.M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of extraction conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts. Food Chemistry, 100, 1356–1364. [3]
The
World
Banana
Forum.
(2015).
Statistics
and
data.
URL
http://www.fao.org/economic/worldbananaforum/statistics/en/. Accessed 14.06.15. [4] González-Montelongo, R., Lobo, M.G., & González, M. (2010). Antioxidant activity in banana peel extracts: Testing extraction conditions and related bioactive compounds. Food Chemistry, 119, 1030-1039. [5] Happi Emaga, T., Robert, C., Ronkart, S.N., Wathelet, B., & Paquot, M. (2008). Dietary fibre components and pectin chemical features of peels during ripening in banana and plantain varieties. Bioresource Technology, 99, 4346-4354. [6] Oliveira, T.I.S., Rosa, M.F., Cavalcante, F.L., Pereira, P.H.F., Moates, G.K., Wellner, N., Mazzetto, S.E., Waldron, K.W., & Azeredo, H.M.C. (2016a). Optimization of pectin extraction from banana peels with citric acid by using response surface methodology. Food Chemistry, 198, 113—118.
20
[7] Tibolla, H., Pelissari, F.M., & Menegalli, F.C. (2014). Cellulose nanofibers produced from banana peel by chemical and enzymatic treatment. LWT – Food Science and Technology, 59, 1311-1318. [8] Pasini Cabello, S.D., Takara, E.A., Marchese, J., & Ochoa, N.A. (2015). Influence of plasticizers in pectin films: Microstructural changes. Materials Chemistry and Physics, 162, 491–497. [9] Penhasi, A., & Meidan, V.M. (2014). Preparation and characterization of in situ ionic cross-linked pectin films: Unique biodegradable polymers. Carbohydrate Polymers, 102, 254–260. [10] Silva, I.S.V., Flauzino Neto, W.P., Silvério, H.A., Pasquini, D., Andrade, M.Z., & Otaguro, H. (2015). Mechanical, thermal and barrier properties of pectin/cellulose nanocrystal nanocomposite films and their effect on the storability of strawberries (Fragaria ananassa). Polymers for Advanced Technologies, doi: 10.1002/pat.3734. [11] Jonoobi, M., Oladi, R., Davoudpour, Y., Oksman, K., Dufresne, A., Hamzeh, Y., &
Davoodi,
R.
(2015).
Different
preparation
methods
and
properties
of
nanostructured cellulose from various natural resources and residues: a review. Cellulose, 22, 935–969. [12] Xu, X., Liu, F., Jiang, L., Zhu, J.Y., Haagenson, D., & Wiesenborn, D.P. (2013). Cellulose nanocrystals vs. cellulose nanofibrils: a comparative study on their microstructures and effects as polymer reinforcing agents. ACS Applied Materials & Interfaces, 5, 2999-3009.
21
[13] Cho, M.-J., & Park, B.-D. (2011). Tensile and thermal properties of nanocellulose-reinforced poly(vinyl alcohol) nanocomposites. Journal of Industrial and Engineering Chemistry, 17, 36-40. [14] Favier, V., Canova, G.R., Cavaillé, J.Y., Chanzy, H., Dufresne, A., & Gauthier, C. (1995). Nanocomposite materials from latex and cellulose whiskers. Polymers for Advanced Technologies, 6, 351-355. [15] Bonilla, J., Talón, E., Atarés, L., Vargas, M., & Chiralt, A. (2013). Effect of the incorporation of antioxidants on physicochemical and antioxidant properties of wheat starch–chitosan films. Journal of Food Engineering, 118, 271-278. [16] Olsson, E., Hedenqvist, M.S., Johansson, C., & Järnström, L. (2013). Influence of citric acid and curing on moisture sorption, diffusion and permeability of starch films. Carbohydrate Polymers, 94, 765-772. [17] Xiaohong, X., & Yang, C.Q. (2000). FTIR spectroscopy study of the formation of cyclic anhydride intermediates of polycarboxylic acids catalyzed by sodium hypophosphite. Textile Research Journal, 70, 64-70. [18] Dastidar, T.G., & Netravali, A.N. (2012). ‘Green’ crosslinking of native starches with malonic acid and their properties. Carbohydrate Polymers, 90, 1620-628. [19] Happi Emaga, T., Ronkart, S.N., Robert, C., Wathelet, B., & Paquot, M. (2008). Characterisation of pectins extracted from banana peels (Musa AAA) under different conditions using an experimental design. Food Chemistry, 108, 463–471.
22
[20] Qiu, L.-p., Zhao, G.-l., Wu, H., Jiang, L., Li, X.-f., & Liu, J.-j. (2010). Investigation of combined effects of independent variables on extraction of pectin from banana peel using response surface methodology. Carbohydrate Polymers, 80, 326–331. [21] Pelissari, F.M., Sobral, P.J.A., & Menegalli,F.C. (2014). Isolation and characterization of cellulose nanofibers from banana peels. Cellulose, 21, 417–432. [22] Khawas, P., & Deka, S.C. (2016). Isolation and characterization of cellulose nanofibers from culinary banana peel using high-intensity ultrasonication combined with chemical treatment. Carbohydrate Polymers, 137, 608–616. [23] Waldron, K.W., & Selvendran, R.R. (1990). Composition of the cell walls of different asparagus (Asparagus officinalis) tissues. Physiologia Plantarum, 80, 568– 575. [24] Manrique, G. D., & Lajolo, F. M. (2002). FT-IR spectroscopy as a tool for measuring degree of methyl esterification in pectins isolated from ripening papaya fruit. Postharvest Biology and Technology, 25, 99–107. [25] Monsoor, M.A., Kalapathy, U., & Proctor, A. (2001). Determination of polygalacturonic acid content in pectin extracts by diffuse reflectance Fourier transform infrared spectroscopy. Food Chemistry, 74, 233-238. [26]
Cranston,
characaterization
E.D., of
&
Gray,
D.G.
polyelectrolyte
(2006).
multilayers
Morphological incorporating
and
optical
nanocrystalline
cellulose. Biomacromolecules, 7, 2522–2530.
23
[27] ASTM (2005) Standard test methods for water vapor transmission of materials. E96-05. In Annual book of ASTM standards. Philadelphia: American Society for Testing and Materials. [28] Ojagh, S.M., Rezaei, M., Razavi, S.H., & Hosseini, S.M.H. (2010). Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chemistry, 122, 161-166. [29] Irissin-Mangata, J., Bauduin, G., Boutevin, B., & Gontard, N. (2001). New plasticizers for wheat gluten films. European Polymer Journal, 37, 1533–1541. [30] Reddy, N., & Yang, Y. (2010). Citric acid cross-linking of starch films. Food Chemistry, 118, 702–711. [31] Seligra, P.G., Jaramillo, C.M., Famá, L., & Goyanes, S. (2016). Biodegradable and non-retrogradable eco-films based on starch–glycerol with citric acid as crosslinking agent. Carbohydrate Polymers, 138, 66–74. [32] 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. [33] Kolodziejski, W., & Klinowski, J. (2002). Kinetics of cross-polarization in solidstate NMR: a guide for chemists. Chemical Reviews, 102, 613-628. [34] Azeredo, H.M.C., Miranda, K.W.E., Rosa, M.F., Nascimento, D.M., & De Moura, M.R. (2012). Edible films from alginate-acerola puree reinforced with cellulose whiskers. LWT – Food Science and Technology, 46, 294–297. 24
[35] Nascimento, D.M., Almeida, J.S., Dias, A.F., Figueirêdo, M.C.B., Morais, J.P.S., Feitosa, J.P.A., & Rosa, M.F. (2014). A novel green approach for the preparation of cellulose nanowhiskers from white coir. Carbohydrate Polymers, 110, 456–463. [36] Morais, J.P.S., Rosa, M.F., Souza Filho, M.S.M., Nascimento, L.D., Nascimento, D.M., & Cassales, A.R. (2013). Extraction and characterization of nanocellulose structures from raw cotton linter. Carbohydrate Polymers, 91, 229–235. [37] Flauzino Neto, W.P., Silvério, H.A., Dantas, N.O., & Pasquini, D. (2013). Extraction and characterization of cellulose nanocrystals from agro-industrial residue – Soy hulls. Industrial Crops and Products, 42, 480–488. [38] Eichhorn, S.J., Dufresne, A., Aranguren, M., Marcovich, N.E., Capadona, J. R., Rowan, S. J., Weder, C., Thielemans, W., Roman, M., Renneckar, S., Gindl, W., Veigel, S., Keckes, J., Yano, H., Abe, K., Nogi, M., Nakagaito, A.N., Mangalam, A., Simonsen, J., Benight, A. S., Bismarck, A., Berglund, L. A., & Peijs, T. (2010). Review:
current
international
research
into
cellulose
nanofibres
and
nanocomposites. Journal of Materials Science, 45, 1–33. [39] Siqueira, G., Bras, J., & Dufresne, A. (2010). Cellulosic bionanocomposites: A review of preparation, properties and applications. Polymers, 2, 728–765. [40] Morelli, C.L., Belgacem, M.N., Branciforti, M.C., Bretas, R.E.S., Crisci, A., & Bras, J. (2016). Supramolecular aromatic interactions to enhance biodegradable film properties through incorporation of functionalized cellulose nanocrystals. Composites: Part A, 83, 80-88.
25
[41] Kargarzadeh, H., Ahmad, I., Abdullah, I., Dufresne, A., Zainudin, S.Y., & Sheltami, R.M. (2012). Effects of hydrolysis conditions on the morphology, crystallinity, and thermal stability of cellulose nanocrystals extracted from kenaf bast fibers. Cellulose, 19, 855–866. [42] Lin, N., & Dufresne, A. (2014). Surface chemistry, morphological analysis and properties of cellulose nanocrystals with gradiented sulfation degrees. Nanoscale, 6, 5384–5393. [43] Moon, J.S., Park, J.H., Lee, T.Y., Kim, Y.W., Yoo, J.B., Park, C.Y., Kim, J.M., & Jin, K.W. (2005). Transparent conductive film based on carbon nanotubes and PEDOT composites. Diamond and Related Materials, 14, 1882—1887. [44] Khan, A., Khan, R.A., Salmieri, S., Le Tien, C., Riedl, B., Bouchard, J., Chauve, G., Tan, V., Kamal, M.R., & Lacroix, M. (2012). Mechanical and barrier properties of nanocrystalline
cellulose
reinforced
chitosan
based
nanocomposite
films.
Carbohydrate Polymers, 90, 1601-1608. [45] Azeredo, H.M.C., Kontou-Vrettou, C., Moates, G.K., Wellner, N., Cross, K., Pereira, P.H.F., & Waldron, K.W. (2015). Wheat straw hemicellulose films as affected by citric acid. Food Hydrocolloids, 50, 1–6. [46] Wang, S., Ren, J., Li, W., Sun, R., & Liu, S. (2014). Properties of polyvinyl alcohol/xylan composite films with citric acid. Carbohydrate Polymers, 103, 94—99. [47] Uranga, J., Leceta, I.. Etxabide, A., Guerrero, P., & de la Caba, K. (2016). Cross-linking of fish gelatins to develop sustainable films with enhanced properties. European Polymer Journal, 78, 82–90. 26
[48] Abdollahi, M., Alboofetileh, M., Behrooz, R., Rezaei, M., & Miraki, R. (2013). Reducing water sensitivity of alginate bio-nanocomposite film using cellulose nanoparticles. International Journal of Biological Macromolecules, 54, 166-173. [49] Huq, T., Salmieri, S., Khan, A., Khan, R.A., Tien, C.L., Riedl, B., Fraschini, C., Bouchard, J., Eribe-Calderon, J., Kamal, M.R., & Lacroix, M. (2012). Nanocrystalline cellulose (NCC) reinforced alginate based biodegradable nanocomposite film. Carbohydrate Polymers, 90, 1757-1763. [50] Oliveira, T.I.S., Zea-Redondo, L., Moates, G.K., Wellner, N., Cross, K., Waldron, K.W., & Azeredo, H.M.C. (2016b). Pomegranate peel pectin films as affected by montmorillonite. Food Chemistry, 198, 107—112. [51] Lorevice, M.V., Otoni, C.G., de Moura, M.R., & Mattoso, L.H.C. (2016). Chitosan nanoparticles on the improvement of thermal, barrier, and mechanical properties of high- and low-methyl pectin films. Food Hydrocolloids, 52, 732–740. [52] Tajeddin, B., Rahman, R.A., & Abdulah L.C. (2010). The effect of polyethylene glycol
on
the
characteristics
of
kenaf
cellulose/low-density
polyethylene
biocomposites. International Journal of Biological Macromolecules, 47, 292—297. [53] Castillo, L., López, O., López, C., Zaritzky, N., García, M.A., Barbosa, S., & Villar, M. (2013). Thermoplastic starch films reinforced with talc nanoparticles. Carbohydrate Polymers, 95, 664—674. [54] Pereda, M., Dufresne, A., Aranguren, M.I., & Marcovich, N.E. (2014). Polyelectrolyte films based on chitosan/olive oil and reinforced with cellulose nanocrystals. Carbohydrate Polymers, 101, 1018–1026. 27
[55] Bardet, R., Belgacem, M.N., & Bras, J. (2013). Different strategies for obtaining high opacity films of MFC with TiO2 pigments. Cellulose, 20, 3025–3037. [56] Donaldson, L.A. (1991). Seasonal changes in lignin distribution during tracheid development in Pinus radiata D. Don. Wood Science and Technology, 25¸15–24. [57] Slavutsky, A.M., & Bertuzzi, M.A. (2014). Water barrier properties of starch films reinforced
with
cellulose
nanocrystals
obtained
from
sugarcane
bagasse.
Carbohydrate Polymers, 110, 53—61. [58] George, J. & Sabapathi, S.N. (2015). Cellulose nanocrystals: synthesis, functional properties, and applications. Nanotechnology, Science and Applications, 8, 45—54. [59] Balaguer, M.P., Gómez-Estaca, J., Gavara, R., & Hernandez-Munoz, P. (2011). Functional properties of bioplastics made from wheat gliadins modified with cinnamaldehyde. Journal of Agricultural and Food Chemistry, 59, 6689–6695. [60] Menzel, C., Olsson, E., Plivelic, T.S., Andersson, R., Johansson, C., Kuktaite, R., Järnström, L., & Koch, K. (2013). Molecular structure of citric acid cross-linked starch films. Carbohydrate Polymers, 96, 270—276. [61] El Miri, N., Abdelouahdi, K., Barakat, A., Zahouily, M., Fihri, A., Solhy, A., & El Achaby, M. (2015). Bio-nanocomposite films reinforced with cellulose nanocrystals: Rheology of film-forming solutions, transparency, water vapor barrier and tensile properties of films. Carbohydrate Polymers, 129, 156–167.
28
[62] Oun, A.A., & Rhim, J.-W. (2015). Preparation and characterization of sodium carboxymethyl
cellulose/cotton
linter
cellulose
nanofibril
composite
films.
Carbohydrate Polymers, 127, 101–109. [63]
ChemicalBook,
CAS
DataBase
List.
Citric
acid.
Available
at
http://www.chemicalbook.com/ChemicalProductProperty_EN_CB9854361.htm, assessed on May 11th, 2016). [64] Synytsya, A., Čopíková, J., Matĕjka, P., & Machovič, V. (2003). Fourier transform Raman and infrared spectroscopy of pectins. Carbohydrate Polymers, 54, 97-106. [65] Yu, J.G., Wang, N., & Ma, X.F. (2005). The effects of citric acid on the properties of thermoplastic starch plasticized by glycerol. Starch/Stärke, 57, 494–504. [66] Peng, X., Mu, T., Zhang, M., Sun, H., Chen, J., & Yu, M. (2016). Effects of pH and high hydrostatic pressure on the structural and rheological properties of sugar beet pectin. Food Hydrocolloids, 60, 161—169. [67] Dawy, M., Shabaka, A.A., & Nada, A.M.A. (1998). Molecular structure and dielectric properties of some treated lignins. Polymer Degradation and Stability, 62, 455-462. [68] Diehl, B.G., Brown, N.R., Frantz, C.W., Lumadue, M.R., & Cannon, F. (2013). Effects of pyrolysis temperature on the chemical composition of refined softwood and hardwood lignins. Carbon, 60, 531–537.
29
[69] Marcott, C., Lo, M., Kjoller, K., Fiat, F., Baghdadli, N., Balooch, G., & Luengo, G.S. (2014). Localization of human hair structural lipids using nanoscale infrared spectroscopy and imaging. Applied Spectroscopy, 68, 564–569. [70] Derenne, A., Claessens, T., Conus, C., & Goormaghtigh, E. (2013). Infrared spectroscopy of membrane lipids. In G. C. K. Roberts (Ed.), Encyclopedia of biophysics (1074—1081). Berlin: Springer. [71] Kloprogge, J.T., Hickey, L., & Frost, R.L. (2004). FT-Raman and FT-IR spectroscopic study of synthetic Mg/Zn/Al-hydrotalcites. Journal of Raman Spectroscopy, 35, 967—974. [72] Le Tien, C., Letendre, M., Ispas-Szabo, P., Mateescu, M.A., Delmas-Patterson, G., Yu, H.L., & Lacroix, M. (2000). [73] Edwards, J.V., Eggleston, G., Yager, D.R., Cohen, I.K., Diegelmann, R.F., & Bopp,
A.F.
(2002).
Design,
preparation
and
assessment
of
citrate-linked
monosaccharide cellulose conjugates with elastase-lowering activity. Carbohydrate Polymers, 50, 305–314. [74] Zhu, X., Liu, B., Zheng, S., & Gao, Y. (2014). Quantitative and structure analysis of pectin in tobacco by
13C
CP/MAS NMR spectroscopy. Analytical Methods, 6,
6407-6413. [75] Wishart, D.S., Tzur, D., Knox, C., Eisner, R., Guo, A.C., Young, N., Cheng, D., Jewell, K., Arndt, D.,Sawhney, S., Fung, C., Nikolai, L., Lewis, M., Coutouly, M.-A., Forsythe, I., Tang, P., Shrivastava, S., Jeroncic, K., Stothard, P., Amegbey, G., Block, D., Hau, D.D., Wagner, J.,
Miniaci, J., Clements, M., Gebremedhin, M., Guo, 30
N., Zhang, Y., Duggan, G.E., MacInnis, G.D., Weljie, A.M., Dowlatabadi, R., Bamforth, F., Clive, D., Greiner, R., Li, L., Marrie, T., Sykes, B.D., Vogel, H.J., & Querengesser, L. (2007). HMDB: the Human metabolome database. Nucleic Acids Research, 35, D521-D526. [76] Tang, H.-R., Wang, Y.-L., & Belton, P.S. (2000). 13C CPMAS studies of plant cell wall materials and model systems using proton relaxation-induced spectral editing techniques. Solid State Nuclear Magnetic Resonance, 15, 239–248. [77] Okushita, K., Komatsu, T., Chikayama, E., & Kikuchi, J. (2012). Statistical approach for solid-state NMR spectra of cellulose derived from a series of variable parameters. Polymer Journal, 44, 895–900.
31
Figure captions: Figure 1. Transmission electron micrograph (TEM) of celulose nanocrystals from banana peels. Figure 2. Film properties as functions of CNC contents and presence/absence of citric acid. Values in the same line (color) followed by the same letter are not significantly different (Tukey, p< 0.05). Values in parentheses are standard deviations. Figure 3. FTIR spectra of pectin films with different cellulose nanocrystals (CNC) and citric acid (CA) contents, as well as of pure pectin and CNC suspension. Films identified as CNC-X-CA-Y, X and Y indicating the cellulose nanocrystal and citric acid contents (%). Figure 4. Scanning electron micrographs from (A) pectin control film; (B) pectin film with 7.5 wt% cellulose nanocrystals; (C) pectin film with 20 wt% citric acid; (D) pectin film with 20 wt% citric acid and 7.5 wt% cellulose nanocrystals. Figure 5.
13C
CP-MAS spectra of films (identified as CNC-X-CA-Y, X and Y
indicating the percent cellulose nanocrystal and citric acid contents, respectively).
32
Fig 1
Fig 2 33
Fig 3
Fig 4
34
Fig 5
35
Table 1. Differences between films without and with citric acid, according to paired t tests for all replicates at all CNC levels.
Averages* Response
t
p
7.92 1.21
-2.88
0.010
4.69 0.84
4.26 0.77
2.40
0.030
Elastic modulus (MPa)
1586 487
1714 452
-1.67
0.115
Opacity (A.nm.mm-1)
2246.3 238.6
2412.0 248.6
-3.56
< 0.01
Insoluble matter (%)
13.13 2.87
14.75 3.91
-3.24
< 0.01
WVP (g.mm.kPa-1.h-1.m-2)
3.31 0.36
3.10 0.33
3.10
< 0.01
Without citric acid
With citric acid
Tensile strength (MPa)
7.36 1.15
Elongation at break (%)
*Averages ( standard deviation) of at least 25 values (5 CNC contents, at least 5 replicates).
Table 2. Contact time (TCH) and proton spin-lattice relaxation times in the rotating frame (1H-T1ρ) of some films.
Film*
TCH (ms)
T1ρ (s)
CNC-0-CA-0
58.6
6.8
CNC-10-CA-0
63.2
7.6
CNC-0-CA-20
85.78
7.8
CNC-10-CA-20
44.47
13.15
* Films identified as CNC-X-CA-Y, X and Y indicating the cellulose nanocrystal and citric acid contents (%).
36