Using gelatin protein to facilitate paper thermoformability

Reactive & Functional Polymers 85 (2014) 175–184

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Using gelatin protein to facilitate paper thermoformability Alexey Khakalo a, Ilari Filpponen a,⇑, Leena-Sisko Johansson a, Alexey Vishtal b, Arcot R. Lokanathan a, Orlando J. Rojas a,c, Janne Laine a a

Aalto University School of Chemical Technology, Department of Forest Products Technology, P.O. Box 16300, 00076 Aalto, Finland VTT Technical Research Centre of Finland, P.O. Box 1603, Koivurannantie 1, Jyväskylä 40101, Finland c North Carolina State University, Departments of Forest Biomaterials and Chemical and Biomolecular Engineering, Raleigh, NC 27695, USA b

a r t i c l e

i n f o

Article history: Received 7 July 2014 Received in revised form 23 September 2014 Accepted 24 September 2014 Available online 2 October 2014 Keywords: Cellulose modification Gelatin Paper formability Paper extensibility Packaging

a b s t r a c t One of the main challenges of fiber-based packaging materials is the relatively poor elongation of cellulose under stress, which limits formability and molding in related products. Therefore, in this investigation we first used cellulose thin films and surface sensitive tools such as quartz crystal microbalance (QCM-D), surface plasmon resonance (SPR) and X-ray photoelectron spectroscopy (XPS) to evaluate the cellulose–gelatin interactions. It was found that the highest adsorption of gelatin onto cellulose occurred at the isoelectric pH of the protein. Based on this and other results, a gelatin loading is proposed to facilitate molecular and surface interactions and, thus to improve the formability of cellulose-based materials in paper molding. Aqueous gelatin solutions were sprayed on the surface of wet webs composed of softwood fibers and the chemical and mechanical changes that occurred were quantified. Upon gelatin treatment the elongation and tensile strength of paper under unrestrained drying was increased by 50% (from 10% to 14%) and by 30% (from 59 to 78 N m/g), respectively. The mechanical performance of gelatin-treated fibers was further improved by glutaraldehyde-assisted cross-linking. The proposed approach represents an inexpensive and facile method to improve the plasticity of fiber networks, which otherwise cannot be processed in the production of packaging materials by direct thermoforming. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Environmental concerns and a more reasonable use of fossil resources have amounted to a growing interest for renewable and biodegradable packaging materials [1]. Cellulose is one of the most abundant biopolymers in the biosphere and the main raw material in production of paper and board [2]. However, one of the main challenges of using cellulose-based packaging materials (such as cups, plates, trays and food containers) is the poor formability of cellulose [3]. Moreover, the barrier properties and moisture resistance of cellulose-based materials are not competitive when compared against traditional plastics. While barrier properties and moisture resistance can be improved by introducing additional coating layers, paperboard formability requires mechanical and chemical modifications of fibers and the fiber network structure to make it feasible. The term ‘‘formability’’ describes the ability of a material to undergo plastic deformation without damage and it is an essential property for 3D-forming processes, where various advanced ⇑ Corresponding author. Tel.: +358 503605623. E-mail address: erkko.filpponen@aalto.fi (I. Filpponen). http://dx.doi.org/10.1016/j.reactfunctpolym.2014.09.024 1381-5148/Ó 2014 Elsevier B.V. All rights reserved.

shapes can be produced. As of today, the most stretchable biobased composite materials have been prepared by reinforcing ductile hydrophobic matrices with natural fibers [4]. However, the interfacial adhesion between the components is not very good and chemical modification of natural fibers is typically required to improve the mechanical properties of such composites [5,6]. It is also important to note here that natural fibers are typically used to a lesser extent when compared to the hydrophobic component of the composites. The main features of paper and paperboard for deep-drawing processes have been recently studied and the ability of a material to deform under applied stress without a failure has been highlighted [3]. The combination of high-consistency wing defibrator refining and subsequent low-consistency valley beating of fibers (laboratory process only) was proposed as a mechanical treatment to improve the extensibility and elongation at break without deteriorating the strength properties of resulting paperboard [7]. Highconsistency treatment decreased the axial stiffness of fibers while the low-consistency refining improved fiber–fiber bonding by strengthening the fibers. It was also reported that the improved bonding between fibers promotes the stretch potential of curly fibers making the paper more extensible [8]. Furthermore, the

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importance of paper drying was also acknowledged, i.e., the shrinkage of paper upon unrestrained drying (negative strain) could be further recovered to increase the overall extensibility of a paper. The extensibility can also be improved by modifying the fiber or fiber network structure. For example, hydroxypropylation of cellulose was used to produce highly extensible and translucent paper [9]. Stretchable paper-based material has been prepared by converting a selectively dissolved fiber surface into the matrix [10,11]. Moreover, the elastic properties of a paper have been improved by applying film forming methylcellulose and lignosulfonate as surface sizing agents [12]. Approximately 50% increase in strain to failure was observed by employing a recombinant cellulose crosslinking protein [13]. Agar has also been used as an additive for improving the extensibility of paper [14]. In addition, the combination of gelatin and nanocellulose substrates has been shown to produce materials with excellent stress performance [15–17]. Gelatin is a mixture of proteins obtained by the hydrolysis of collagen, the most abundant protein in waste products like skins, connective tissues, bones and cartilage of predominant bovine animals. When heated to 40 °C, gelatin dissolves in water with a formation of random coiled chains. Upon cooling, gelatin chains partially recover the original triple-helix structure of collagen. Thus, gelatin gels form an ensemble of physically interconnected triple helices, which are held together by intermolecular hydrogen bonds. From the point of view of its chemical structure, gelatin is a weak polyampholyte. Typically, approximately 13% of gelatin backbone is positively charged (lysine and arginine), 12% is negatively charged (glutamic and aspartic acid) and 11% is hydrophobic in nature (leucine, isoleucine, methionine and valine) [18]. Gelatin is widely utilized in the food industry. In pharmaceutical field gelatin is used in controlled delivery systems [19,20] and for fabrication of three-dimensional gelatin-based polymer scaffolds for tissue-engineering and wound dressing purposes [21–23]. Moreover, gelatin-based films and interpenetrating polymeric networks have been proposed as alternatives to synthetic food packaging materials [2,16,24]. Apart from the aforementioned advantages of gelatin, there are some drawbacks that hinder its applications, among which poor mechanical properties and moisture sensitivity are the most limiting ones. Different approaches to overcome these drawbacks have been proposed but chemical cross-linking is by far the most effective method to improve mechanical, thermal, and water-resistance properties of gelatin. A variety of chemicals, biomolecules and enzymes have been used for cross-linking, such as glutaraldehyde [18,19], epoxides [25], glyoxal [26], isocyanates [27], formaldehyde [25,26], glyceraldehyde [28], genipin [29] and transglutaminase [30]. However, glutaraldehyde is the most widely used due to its commercial availability, short-reaction time and low cost. In addition, it has an excellent ability to stabilize collagenous materials and therefore to improve the strength properties and water resistance [18]. Gelatin has been widely studied for its gel and film forming abilities in combination with cross-linker, like glutaraldehyde, or with plasticizer, like glycerol and sorbitol. However, despite the growing interest, to our knowledge there are no reports available on the application of gelatin for production of highly extensible paper and paperboard. In this communication, we first unveil the nature of cellulose–gelatin interactions by using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) to then apply aqueous gelatin solution via spraying on cellulose fiber networks. As a result, an improved formability of paper was achieved. The morphology and composition of adsorbed gelatin layer were analyzed by scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray

photoelectron spectroscopy (XPS), all of which provide supporting evidence of the mechanism and mode of action of gelatin as an inexpensive and facile method to endow fiber networks with plasticity to make them suitable for 3D thermal forming. 2. Experimental 2.1. Materials First thinning bleached softwood kraft fiber sheets (cellulose 81.7%, xylan 9.2%, glucomannan 9.0% and total lignin <0.5%) were provided by Pietarsaari mill, UPM-Kymmene. Gelatin from porcine skin (Type A, 300 g Bloom gel strength, #232-554-6), glutaraldehyde solution (50 wt.% in H2O, #340855) and polystyrene (280 kDa molecular weight, #182427) were obtained from Sigma–Aldrich (US). Trimethylsilyl cellulose (TMSC) was synthetized as described elsewhere [31]. 2.2. Preparation of cellulose surfaces Cellulose-gelatin interactions were investigated by using goldcoated sensors in SPR and QCM-D experiments. The sensors (SPR gold chips or QCM crystals) were first cleaned with UV/ozone treatment for 15 min followed by spin coating with 0.1 wt.% polystyrene in toluene (4000 rpm, 60 s). Prepared polystyrene-coated sensors were then dried in an oven at 60 °C for 10 min to ensure a uniform hydrophobic layer suitable for trimethylsilyl cellulose (TMSC) deposition. TMSC was deposited on the polystyrene-coated sensors by using the Langmuir–Schaeffer (LS) lifting deposition technique as described by Tammelin et al. [32]. The TMSC layer was then converted to cellulose via desilylation with hydrochloric acid vapor as described elsewhere [33]. The crystallinity degree, thickness, and roughness of the LS-cellulose films prepared in the same manner have previously been observed to be 54%, 17.8 nm, and 0.5 nm, respectively [34]. Before QCM-D and SPR experiments the cellulose films were allowed to stabilize overnight in the appropriate buffer solution. 2.3. Adsorption experiments using cellulose sensors Prior to QCM-D and SPR adsorption experiments, gelatin was dissolved in Milli-Q water and dialyzed using a 10–12 kDa mesh membrane tube (SpectraPor, Spectrumlabs) and freeze-dried. Isoelectric point of gelatin was measured using zeta-potential analyzer (Malvern Zetasizer Nano ZS device, Malvern Instruments, Malvern, UK). Dialyzed gelatin was dissolved in 10 mM acetate, phosphate and bicarbonate buffers at pH 4, 5.8 and 10, respectively at 45 °C for 30 min to yield 0.1 mg/mL concentration. Afterwards, gelatin solution was filtered using 0.45 lm filters and degassed. Both QCM-D and SPR experiments were performed at a constant flow rate of 100 lL/min and the temperature was maintained at 21 °C until adsorption plateau was reached. Thereafter, rinsing with polymer-free buffer solution was applied to ascertain the irreversible binding of gelatin to cellulose surfaces pre-adsorbed on the (QCM/SPR) sensors. Finally, the sensors were washed with Milli-Q water and stored in desiccator until further investigation. Each set of experiments was performed at least two times. 2.4. Adsorbed mass by using quartz crystal microbalance and surface plasmon resonance Gelatin adsorption onto cellulose and properties of adsorbed gelatin layer were investigated by using quartz crystal microbalance with dissipation (QCM-D) monitoring (E4 instrument, QSense AB, Sweden) and surface plasmon resonance (SPR) unit

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(SPR Model Navi 200, Oy BioNavis Ltd., Tampere, Finland). With QCM-D, changes in mass and film viscoelastic properties on the sensor surface are detected by simultaneous monitoring changes in frequency (Df) and dissipation (DD), respectively, as a function of time at the fundamental resonance frequency (5 MHz) and its overtones (15, 25, 35, 45, 55, and 75 MHz). The principles of QCM-D operation and data analysis can be found elsewhere [35,36]. If the adsorbed layer is rigid, uniformly distributed on the surface, and small compared to the mass of the solid support (crystal), the Sauerbrey’s relation can be applied to calculate the mass change upon adsorption (Dm) [37]:

Dm ¼

c  Df n

ð1Þ

where C = 17.7 ng/cm2 for 5 MHz crystal, Df is change in frequency, and n is the overtone number. However, if the adsorbed layer is soft or highly hydrated, the Voigt viscoelastic model (calculations by QTools software, version 2.1, Q-Sense, Västra Frölunda, Sweden) is applicable. This model utilizes frequency and dissipation data from several overtones and can be represented as a system consisting of a spring and a dashpot filled with viscous fluid connected in parallel [38]. The density of the adsorbed gelatin layer in this latter case was assumed to be 1200 g/m3. Frequency shifts (Df) at the third overtone (15 MHz) are presented since this overtone usually has the best signal-to-noise ratio. In SPR experiments the so-called SPR angle can be used to derive the thickness of adsorbed layer: [39]

d¼

ld DSPR angle  2 mðna  n0 Þ

(Valley beater) consistency refining until the degree of refining measured by Schopper–Riegler (SR) was 25 [7]. Next, the fibers were washed to Na-form in order to remove adsorbed metal ions and water-soluble substances, according to a procedure described by Laine et al. [42]. Handsheets were prepared using deionized water according to ISO 5269-1 except that a grammage of 200 g/m2 was used. Gelatin was swelled in deionized water for 1 h and then dissolved at 45 °C for 30 min. Gelatin solution (4 wt.%) was used for spraying by using a universal spray gun (Wagner W 140P, J. Wagner GmbH, Germany). For the preparation of gelatin-treated paper containing cross-linker, glutaraldehyde solution was sprayed on top of the sprayed gelatin layer. Finally, samples were air-dried (23 °C and 50% RH) between two plastic wire nets, which enabled the handsheets to shrink freely without excessive curling. All the characterizations were conducted for the freely dried samples, unless otherwise stated. 2.7. Tensile testing The tensile strength and elongation at break of the gelatintreated papers were measured with a MTS 400/M (MTS Systems, USA) vertical tensile tester with a load cell of 2 kN equipped with TestWorks 4.02 measuring program and run according to SCAN-P38. At least 20 replicate specimens from each gelatintreated paper type were measured. 2.8. Sheet formability

ð2Þ

where DSPR angle is a change in the SPR angle, ld is a characteristic evanescent electromagnetic field decay length (240 nm), estimated as 0.37 of the light wavelength; m is a sensitivity factor for the sensor (109.94°/RIU) obtained after calibration of the SPR, n0 is the refractive index of the bulk solution (1.33 RIU) and na is the refractive index of the adsorbed layer (assumed value of 1.60 RIU measured for crystalline proteins). The surface excess concentration was calculated according to Eq. (3):

M ¼dv

177

ð3Þ

where d is the calculated thickness of the adsorbed layer and m is the specific density of the layer (1.35 g/cm3) [40]. 2.5. Surface topography by atomic force microscopy (AFM) Topographical changes on the cellulose surfaces after gelatin adsorption were analyzed by AFM equipped with Nanoscope IIIa Multimode scanning probe microscope from Digital Instruments Inc. (Santa Barbara, CA, USA). 1  1 lm2 images were recorded by using silicon cantilevers in air scanned via the tapping mode. Prior to measurements, the samples were allowed to dry in desiccator at room temperature at least overnight. At least three different regions on each sample were analyzed. Image analyses were performed using NanoScope Analysis 1.2 software, no image processing except flattening was done. The rms surface roughness was measured from 1  1 lm2 scan sizes.

Formability strain and strength were measured using a 2D formability tester developed by VTT. This unit is equipped with a double-curved heated press (temperatures up to 250 °C), blank holders and an IR sensor (OmegaÒ OS36) to measure the temperature of the paper. Typically, a paper with a grammage range from 80 to 250 g/m2 can be preheated to the die temperature within 0.5–0.7 s. In practise this means that the temperature of the paper at the moment of forming is close to that of the die. Fig. 1 presents the basic operation principle of 2D formability tester and a photographic image of the formed paper strip. The forming proceeds as follows: a paperboard sample (15 mm wide  65 mm long) is fixed by the two blank holders. The press is then moved into contact with the sample and retained still for 0.5 s in order to preheat the sample. Then, press continues a downward movement until breakage of the sample. The velocity of the forming press was 10 mm/s [43]. The formability strain of the samples was measured as an average value collected from 10 samples at die temperatures of 23 (room temperature), 45, 60, 75, 90, 105, 120

2.6. Gelatin-modified paper Gelatin-modified paper was prepared by repeated spraying of aqueous 4 wt.% gelatin solution (with respect to dry cellulose fibers) on freshly prepared cellulosic fiber handsheets before wet pressing, as described in Ref. [41]. Samples with different gelatin loading were obtained. Prior to handsheet preparation the fibers were pre-treated combining high (Wing defibrator) and low

Fig. 1. Schematic representation of 2D formability tester and formed strip of paper.

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and 135 °C. In order to evaluate influence of the moisture content on the paper formability, the paper samples were conditioned at two different relative humidity levels 50% and 75% (23 °C) prior to forming. 2.9. Fiber chemical changes by Fourier transformed infrared spectroscopy (FTIR) Bulk chemical characteristics and bonding of both cross-linked and non-cross-linked gelatin-treated paper, as well as reference samples were investigated by an Avatar 360 FTIR spectrometer (Thermo Nicolet). Spectra were recorded at room temperature in absorbance mode within the wavenumber range of 400– 4000 cm1 at 4 cm1 resolution using KBr pellets. Each set of data acquisition was performed at least two times, for each spectrum the number of scans was set to 32 and CO2 and H2O corrections were carried out prior to every measurement. Omnic 6.0a software was used for data acquisition and analysis. 2.10. Imaging by scanning electron microscopy (SEM) A FEI Quanta 200 scanning electron microscope (SEM) was utilized for in plane and cross section imaging. The SEM was operated in back scattered electron mode at an emission current of roughly 100 lA, and using an accelerating voltage of 12.5 kV or 15 kV. The working distance was set to 10 mm and a spot size of 5–6 was used. The samples were previously coated with evaporated carbon using a BALZERS SCD 050 sputter coater equipped with a nonrotating base. The cross sections were prepared by a Hitachi IM4000 cross section cutter prior to the SEM investigation. 2.11. Surface chemical composition via X-ray photoelectron spectroscopy (XPS) Surface chemical composition of fibers as well as QCM surfaces was investigated with XPS Kratos Axis Ultra instrument (Kratos Ltd., Manchester, UK). The samples were measured after an overnight pre-evacuation using monochromated Al Ka irradiation at 100 W (8 mA, 12.5 kV) under neutralization. Wide scan spectra were measured using 1 eV step and 80 eV pass energy, and the high resolution measurements were recorded with 0.1 eV step and 20 eV pass energy. Experimental conditions were monitored with an in-situ reference (100% cellulose filter paper). All spectra were collected at an electron takeoff angle of 90°. Each sample was analyzed on at least 3 different locations and the mean of three values are presented. The area and depth of analysis was 1 mm2 and less than 10 nm, respectively. No sample degradation due to ultrahigh vacuum or X-ray radiation was observed during the XPS measurements. Both elemental wide-region data and high resolution spectra of carbon (C 1s) and oxygen (O 1s) were collected. The relative amounts of carbon, oxygen, nitrogen (protein marker), and silicon were determined from C 1s, O 1s, N 1s, and Si (Si 2p or Si 2s or 1s) signals from low-resolution scans, and the high-resolution C1s spectra were curve fitted for further chemical analysis, using parameters defined for cellulosic materials [44]. 2.12. Stamping of gelatin-treated paper Stamping of prepared gelatin-treated papers was conducted at the laboratory scale using 3D forming device (Stora Enso RC, Imatra). The forming conditions were as follows: temperature of the cavity 140 °C, speed of the die 50 mm/s, blank holding force 3 kN, moisture content of paper ca. 8% (conditioned at 50% RH and 23 °C).

3. Results and discussions 3.1. Adsorption of gelatin on cellulose Cellulose–gelatin interactions were studied by using Langmuir– Schaefer (LS) cellulose films [32]. The LS cellulose films are made up of pure cellulose and have been taken as a good surface chemistry model of cellulose fibers. LS films are typically very smooth and the surface quality can be tuned because the molecular transfer ratio (and resulting degree of molecular packing) can be regulated during the deposition stage. Moreover, the absence of voids and lower surface roughness of LS films simplify the interpretation of data from adsorption and interfacial interactions. Interactions between slightly negatively charged cellulose LS films and gelatin were studied with QCM-D and SPR under identical experimental conditions. It should be noted here that the electrostatic charge of gelatin is dependent on the solution pH, i.e., gelatin is negatively charged above its isoelectric pH of 5.8 and positively charged below it. Gelatin adsorption on cellulose LS films at pH 4, 5.8 and 10 is presented in Fig. 2. It was found that the gelatin adsorption is rather irreversible as no significant desorption was observed after rinsing with the background buffer solution. Moreover, the highest adsorbed amount was detected at pH 5.8 which is the isoelectric pH of gelatin. This is in line with the well-known fact that protein adsorption is maximized near the isoelectric pH [45]. It has also been speculated that the gelatin adsorption is enhanced by its flexible configuration at the interface so that the surface of cellulose can accommodate a large amount of protein molecules, i.e., by suitably changing their structural orientation at the polysaccharide interface [46]. Similar observations have been reported for the gelatin adsorption on noncellulosic hydrophobic surfaces such as phosphatidylcholine coated silica [47]. At pH 4 gelatin is positively charged and it behaves as a polyelectrolyte (pKa of glutamic COOH is 4.3). Therefore, it was expected for the adsorption of gelatin onto slightly negatively charged cellulose to be high considering the electrostatic interactions. However, gelatin adsorption at pH 4 was found to be less than at pH 10, when gelatin is negatively charged (pKa of arginine is 9.0). The amount of free amino groups in gelatin molecules is much higher than carboxylic acid groups, therefore, if compared to observations at pH 4, there are more unprotonated e-amino groups than protonated carboxylic acid groups at pH 10 [18]. Overall, there is indication that hydrogen bonding and other non-specific interactions are important in cellulose-gelatin interactions. The thicknesses of the adsorbed gelatin layers determined by QCM-D were found to be significantly higher than those obtained from SPR experiments. This confirms the hydrogel-like nature of gelatin in aqueous solution. In fact, the amount of coupled water in the adsorbed protein can be calculated by comparing the QCM-D and SPR data since difference in the calculated adsorbed mass corresponds to such contribution. The water content of gelatin adsorbed at the isoelectric pH is slightly lower (92%) when compared to those at pH 4 and 10 (96% in both cases). This is likely attributed to the reduced amount of charged groups in gelatin at the isoelectric pH, which decreases water coupling or hydration. The respective adsorbed masses for gelatin were calculated by applying Voigt viscoelastic model and SPR mass (Eqs. (2) and (3)) since the adsorbed gelatin layers did not meet the Sauerbrey’s conditions (see Table 1). The LS cellulose films with adsorbed gelatin were further investigated with X-ray photoelectron spectroscopy (XPS). Fig. 2c includes the oxygen/nitrogen atom % ratios (O/N) of respective surfaces. As expected, the highest amount of nitrogen (lowest O/N ratio) was found from the surface in which the gelatin was adsorbed at pH 5.8. XPS results were in good agreement with the adsorption studies (QCM-D and SPR).

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Fig. 2. Adsorption of gelatin on LS cellulose films measured as a shift in QCM frequency (third overtone, Df) (a), by SPR (reported as change in SPR angle) and resulting changes in surface chemical composition from XPS data (c) as indicated by the ratios of oxygen and nitrogen atom concentration on the surface of the cellulose surfaces after gelatin adsorption at three different pH values. The O/N ratio of gelatin is given as a reference.

Table 1 Calculated QCM-D and SPR adsorbed masses (Dm, mg/m2), layer thicknesses (nm), and coupled water of gelatin adsorbed from aqueous solutions on LS cellulose surfaces. The QCM adsorbed mass was calculated by using the Voigt viscoelastic model. QCM-D (wet weight)

pH 4 pH 5.8 pH 10

SPR (dry weight)

Water (%)

Thickness (nm)

Dm (mg/m2)

Thickness (nm)

Dm (mg/m2)

7.75 ± 0.05 21.64 ± 0.14 12.77 ± 0.1

9.3 ± 0.2 26 ± 0.8 15.3 ± 0.4

0.28 ± 0.05 1.68 ± 0.1 0.48 ± 0.05

0.38 ± 0.05 2.27 ± 0.1 0.65 ± 0.05

Finally, topographical changes of gelatin-modified LS cellulose surfaces were studied with atomic force microscopy (AFM). At pH 5.8, the adsorbed gelatin layer displays globular conformation, whereas at pH 4 and pH 10 the layer appears to contain more prominent, elongated structures (Fig. 3). In addition, a higher roughness is found for the samples obtained after adsorption at pH 4 and pH 10 (0.764 and 0.817, respectively) compared to that found at pH 5.8 (0.599). These observations can be ascribed to the reduced solution stability of gelatin molecules at isoelectric pH while the large degree of gelatin swelling at pH 4 and 10 is a contributing factor to the increased surface roughness under these conditions. Based on the QCM and SPR results, it is hypothesized that the addition of gelatin, its effect on hydrogen bonding and interaction via amino acids and hydrophobic effects, could be contributing factors to achieve enhanced fiber extensibility. Therefore, the following sections summarize the effect of gelatin when applied on the surface of fibers, under optimal conditions to maximize adsorption.

96 91 96

3.2. Mechanical performance of gelatin-modified paper Gelatin-modified paper was obtained by repeated spraying of aqueous 4 wt.% gelatin solution (with respect to dry cellulose fibers) on freshly prepared handsheets before wet pressing. Samples with different gelatin loading were obtained. Typical stress– strain curves of the gelatin-modified paper are presented in Fig. 4a and a summary of the key mechanical properties are reported in Table 2. Gelatin upon drying tends to recover partial collagen-like triplehelix structure stabilized mainly by the formation of inter-chain hydrogen bonds between carbonyl and amine groups. Thus, the films containing only gelatin are brittle and susceptible to crack due to the strong cohesive energy density of the polymer [24]. However, the application of gelatin as a minor component in a cellulosic composite may contribute to the fiber–fiber bonding by filling the voids and by increasing the contact areas between the fibers. As evident from Table 2, tensile strength of the gelatin-modified paper increased until 8 wt.% gelatin content, indicating that

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Fig. 3. AFM height images and corresponding roughness profiles of unmodified cellulose (a), and cellulose with adsorbed gelatin at pH 4 (b), 5.8 (c) and 10 (d). The Z-range of all the images is 5 nm.

Fig. 4. Stress–strain curves of gelatin-modified paper (a) and glutaraldehyde cross-linked gelatin-modified paper (b).

Table 2 Mechanical properties of gelatin-modified paper and glutaraldehyde cross-linked gelatin-modified paper. Sample

Density (kg/m3)

Strain to failure (%)

Tensile index (N m/g)

Stiffness index (kN m/g)

TEA index (J/g)

Gelatin addition (wt.%) 0 740 4 770 8 820 12 760 16 680 20 710

9.7 ± 0.5 11.4 ± 0.4 14.3 ± 0.6 18.0 ± 1.2 21.1 ± 1.4 21.6 ± 2.0

59.3 ± 3.1 70.3 ± 4.4 77.9 ± 5.6 74.2 ± 4.2 73.2 ± 5.8 70.2 ± 5.3

2.6 ± 0.3 3.0 ± 0.4 3.4 ± 0.3 2.7 ± 0.4 2.5 ± 0.3 2.3 ± 0.4

3.3 ± 0.2 4.8 ± 0.4 6.3 ± 0.5 7.7 ± 0.7 9.3 ± 0.7 9.3 ± 0.8

4 wt.% gelatin addition + glutaraldehyde 4 + 0.5 730 4 + 1.0 740 4 + 1.5 730

14.1 ± 0.6 15.0 ± 0.8 14.3 ± 0.7

77.0 ± 5.9 76.8 ± 5.8 73.3 ± 5.7

3.0 ± 0.4 3.0 ± 0.4 3.0 ± 0.4

6.4 ± 0.3 6.7 ± 0.5 6.2 ± 0.5

an optimal cellulose–gelatin interaction is reached at this particular loading level. Moreover, the density of 8 wt.% gelatin-containing paper increased from 740 (reference sample) to 820 kg/m3, which is still in the range of typical densities of commercial paperboards utilized for packaging purposes [48].

The strength properties of the treated paper slightly decreased at gelatin loadings higher than 8 wt.%. However, the extensibility (strain to failure) and TEA Index (the tensile energy absorbed by the sample before failure) of the gelatin-modified paper is found to increase steadily with gelatin within the whole range of loading.

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181

Fig. 5. Formability strain (a and c) and strength (b and d) as a function of processing temperature of paper as well as gelatin-treated paper with and without crosslinking. Samples were conditioned at relative humidity levels of 50% (a–b) and 75% (c–d). x denotes the water content of the samples.

Fig. 6. SEM micrographs of reference sample (a and d), gelatin-treated paper (4 wt.% gelatin) without (b and e) and with crosslinking (1 wt.% of glutaraldehyde) (c and f). Plane view images are shown in panels a–c and the respective cross-section are included in panels d–f.

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It is important to note that gelatin application facilitates drying shrinkage of the treated paper. This was observed by comparing the extensibilities of samples that were dried under restraint. For instance, while dried under restraint, the extensibility of the sample treated with 4% gelatin (GEL 4%) was 2% lower than that of the sample subjected to free drying. Furthermore, the shrinkage was more pronounced for the samples with higher gelatin content (data not shown). Therefore, it is reasonable to postulate that the increased extensibility with gelatin loadings higher that 8 wt.% is mainly attributed to the shrinkage of the treated paper. According to Seth [8], paper extensibility generally depends on the stretch-potential of fibers, the degree of bonding between fibers and the structure of the network. This is in line with our findings which indicate that gelatin improves the fiber bonding (enhanced tensile properties) and also contributes to the paper structure (facilitates paper shrinkage during drying). Moreover, the load–elongation curves of gelatin-modified paper indicate a much more plastic behavior than the reference paper (control sample with no gelatin treatment). It is likely that the applied stress is more evenly distributed in the cellulose–gelatin network, which in turn allows the axially deformed cellulose fibers to release their extension potential. Fig. 4b illustrates the stress–strain curves of gelatin-treated paper after glutaraldehyde cross-linking (see also Table 2). The elongation at break of the treated papers increased from 11.4% to 14.1% after crosslinking with 0.5% glutaraldehyde (w/w based on cellulose). Likewise, the tensile index increased from 70.3 to 77 N m/g. At 1% glutaraldehyde addition level the strain to failure reached maximum value of 15%, whereas the tensile strength remained almost unchanged when compared to that of 0.5% glutaraldehyde addition. However, further addition of the cross-linker (1.5%) did not improve the mechanical properties of the treated papers. It should be also noted that the tensile stiffness of gelatin-treated paper increased approximately 30% reaching a maximum value of 3.4 kN m/g upon 8 wt.% gelatin addition (Table 2). For the comparison, the tensile stiffness index of the untreated reference sample was found to be 2.6 kN m/g. Surprisingly, the stiffness properties remained unchanged upon cross-linker application. Glutaraldehyde is the most commonly used cross-linker with proteins and polymers containing amine functional groups. It is widely accepted that glutaraldehyde reacts with the residues of amino acids, particularly with the unprotonated e-NH2 functional

groups of lysine and hydroxylysine and the amino groups of the N-terminal amino acids. Therefore, glutaraldehyde forms bonds similar to those of Schiff bases [40]. Typically, cross-linking of gelatin films enhances their rigidity and tensile strength but it is also produces a dramatic reduction in the extensibility, probably due to the depression of molecular mobility [49]. Interestingly, in the case of the systems discussed here the introduction of glutaraldehyde improved the elongation. This observation can be rationalized by the enhanced intermolecular bonding of fibers carrying adsorbed gelatin. Therefore, the stretch capacity of the individual cellulose fiber becomes more pronounced. In fact, it was found that the extensibility correlated strongly with the strength of the material (Table 2). 3.3. Formability The formability of gelatin-treated paper was examined as a function of the forming die temperature. The formability strain and strength of gelatin-treated paper at two different humidity levels (50% and 75%) are presented in Fig. 5. With the increased processing temperatures the elongation of the gelatin-treated papers was generally increased until reaching a maximum (Fig. 5a and c). It is likely that the elevated temperature reduces intermolecular forces within the structure, which increases the mobility of polymeric chains and improves their flexibility [50]. However, the strength of the samples was reduced throughout the investigated temperature range (Fig. 5b and d). It was also observed that further increase in temperature decreases the formability strain which may be due to the extensive softening of wood polymers leading to the immature initiation of a fracture. The maximum increase in elongation with temperature was approximately 2.5% regardless of the water content of the gelatin-treated paper (with and without cross-linker, Fig. 5a and c). It should be noted that the elongation also increased in the case of the reference paper samples but to a lesser extent (increase of 1.8% and 0.85%). Therefore, it can be postulated that the disruption of hydrogen bonding between cellulosic fibers, facilitated by entrapped water, is more pronounced in the reference paper samples compared to the gelatin-treated paper. The humidity level, which correlates with the water content in the sample, was found to have a minor effect in the elongation of the samples, i.e., the gelatin-treated paper with higher water content only exhibited approximately 1% larger strain compared to the samples with lower moisture levels. This finding may be attributed to the plasticizing effect of water. However, it should be noted that the maximum elongation values for gelatin-treated papers at different moisture content were found at different processing temperatures (Fig. 5a and c). In general, higher water content shifted the maximum elongation values to lower temperatures. This observation is in a good agreement with the data published by Yakimets et al. for gelatin films. They reported that by increasing the water content from 7% to 11% the glass transition temperature of the gelatin films decreased from 95 to 75 °C [51]. 3.4. Morphology (SEM) of gelatin-treated fibers

Fig. 7. FTIR spectra of cross-linked (bottom profile) and non-cross-linked (middle profile) gelatin-treated papers. FTIR spectrum of cellulose is shown as a reference (upper profile).

The morphology and microstructure of the gelatin-treated papers was investigated by SEM (Fig. 6). As expected, the mechanical refining resulted in dislocations, cracks and microcompressions throughout the fiber mat (Fig. 6a) [7]. However, despite the protein’s ability to form continuous matrices, the surface morphology of the gelatin-treated papers remained intact upon gelatin application (Fig. 6b and c). Fig. 6e and f illustrates cross-section SEM micrographs of the gelatin-treated papers. It is apparent that despite its surface application, gelatin does not accumulate on the surface of the paper;

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Table 3 Surface elemental composition of unmodified, gelatin-modified and cross-linked gelatin-modified paper. GLU 1% stands for 1 wt.% glutaraldehyde treated paper, GEL 4% is sample sprayed with 4 wt.% gelatin solution and GEL 4% + GLU 1% is 4 wt.% gelatin containing sample treated with 1 wt.% glutaraldehyde solution. Sample

Atomic concentrations, % C 1s

Reference GLU 1% GEL 4% GEL 4% + GLU 1%

60.4 ± 0.2 60.4 ± 0.2 65.0 ± 0.2 66.7 ± 0.2

High resolution C survey C 1s spectra

N 1s

O 1s

CAC

CAO

C@O

COO

10.8 ± 0.1 15.7 ± 0.2

39.6 ± 0.2 39.6 ± 0.2 24.1 ± 0.2 17.3 ± 0.1

4.4 ± 0.1 5.1 ± 0.1 28.0 ± 0.2 38.7 ± 0.2

75.1 ± 0.2 73.2 ± 0.2 47.4 ± 0.2 34.1 ± 0.1

18.8 ± 0.1 19.2 ± 0.2 22.4 ± 0.1 24.3 ± 0.1

1.7 ± 0.1 2.5 ± 0.1 2.2 ± 0.1 2.9 ± 0.1

Fig. 8. Stamped unmodified paper (a) and gelatin-treated paper (4 wt.% gelatin) (b). The dimensions of the formed shapes were approximately 110 mm (length)  70 mm (width)  35 mm (depth).

instead, it partially penetrates throughout the fiber network. Gelatin fills the voids between cellulosic fibers (Fig. 6e) and improves the bonding within the system. Moreover, this leads to a more even distribution of the applied stress, which favors the mechanical properties of the paper. However, when glutaraldehyde crosslinking is performed, gelatin penetration inside the fiber web is slightly deteriorated (Fig. 6f) possibly due to a fast rate of cross-linking that locks the protein in place. Thus, the surface effect is more pronounced in this case.

3.5. Bulk chemical characterization (FTIR) of gelatin-treated papers The results pointed out thus far indicate that gelatin can be adsorbed onto cellulose fibers and the gelatin-modified papers have improved mechanical properties. Moreover, the mechanical properties of gelatin-treated paper can be further improved by introducing a cross-linking agent (glutaraldehyde). This indicates successful crosslinking reaction between glutaraldehyde and the gelatin-treated fibers. For example, Bigi et al. reported that the glutaraldehyde assisted crosslinking of the gelatin films increased their stiffness (up to 25-fold increase in the initial Young’s modulus) [52]. It is likely that the crosslinking proceeds via Schiff base (imine) formation between glutaraldehyde and the unprotonated e-amino groups of gelatin-treated fibers [53]. Moreover, it is possible that in gelatin-treated paper these linkages (imines) act as energy dissipation centers which may contribute to the total extensibility of the system. FTIR was used to investigate the nature of the chemical bonds within the gelatin-treated paper. FTIR spectra of reference cellulose paper and gelatin-treated paper with and without cross-linker are presented in Fig. 7. As expected, the spectra appeared rather similar because of the low gelatin and glutaraldehyde loading. However, a signal at 1540 cm1 is only found for gelatin-containing samples and is attributed to amide II bending vibrations of the protein molecules. Moreover, the increased intensity of the band at about 1640–1650 cm1 (when compared to the reference spectrum) indicates the presence of amide I bending vibrations of gelatin. However, it should be noted that the interpretation of this spectral region is very challenging because of the overlapping peak from the absorbed water which is present in all the samples.

A more pronounced peak upon spectra magnification was observed at 1640 cm1, which could possibly be attributed to the C@N stretching vibration of the imine group of Schiff bases, a structure, which should only occur after successful crosslinking of amine groups of gelatin and glutaraldehyde (Fig. 7, bottom spectra). Moreover, upon magnification it is possible to observe a small peak at 916 cm1 that is attributed to the bending vibrations of monosubstituted alkenes which could be formed by glutaraldehyde cross-linking of gelatin molecules [54]. FTIR demonstrated the presence (retention) of gelatin in the treated papers, which supports the mechanical properties findings discussed before. Moreover, there is an indication of imine formation between glutaraldehyde and gelatin-treated cellulose fibers, which may explain the improved mechanical performance of the cross-linked samples. 3.6. Surface chemical analysis (XPS) of gelatin-treated paper Table 3 summarizes the XPS results of untreated and gelatintreated papers with and without crosslinking. In addition, the surface of glutaraldehyde-treated paper was investigated in order to better access the chemical changes in the surface of cross-linked gelatin-modified paper. The surface elemental compositions of unmodified and glutaraldehyde-modified paper were similar and the details in the high resolution carbon spectra indicated that glutaraldehyde is not concentrated on the surface of the paper substrate but rather diffuses inside the fiber, facilitated by its low molecular weight and weak affinity to cellulose (REF vs GLU 1%, Table 3). The amount of CAC bonds significantly increased (from 28% to 39%) as a result of glutaraldehyde addition. This observation may be attributed to the cross-linking reaction, which leads to the formation of new CAC bonds. Moreover, the amount of nitrogen which serves as a protein marker is higher for cross-linked paper than for the non-crosslinked sample (16% and 11%, respectively). This indicates that glutaraldehyde promotes gelatin retention inside the fiber web after wet pressing. 3.7. Stamping of the gelatin-treated paper Overall, the results presented thus far indicate that gelatin can be used to improve the strength and extensibility of paper.

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Therefore, gelatin treatment can be used to produce advanced 3Dshapes, for example, by directly stamping paper, without need of pre-creasing. As such, paper samples were thermoformed by the stamping method. The resultant thermoformed material, in the shape of a trough, indicated a superior performance of the system after gelatin treatment (Fig. 8). In fact, with no gelatin addition it is not possible to directly process paper by this method. The processability of gelatin-treated fibers is expected to also facilitate other types of forming processes.

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4. Conclusions

[25]

We demonstrate the potential of gelatin treatment in 3D-forming processes for packaging applications. First, QCM-D and SPR studies with cellulose films indicated a maximum adsorption of gelatin at the isoelectric pH of 5.8, which was further demonstrated by using AFM and XPS. Conditions for maximum gelatin adsorption were employed by spraying protein solution onto paper sheets (up to 8 wt.% gelatin content), which was noted to improve their extensibility and strength properties. The plastic behavior was further improved at higher gelatin loadings (over 8 wt.%) but with a reduced strength at failure. Glutaraldehyde-assisted crosslinking of gelatin-treated fibers further enhanced the mechanical performance of the material. For instance, by adding 1 wt.% of glutaraldehyde to paper loaded with 4 wt.% gelatin increased the elongation from 11% to 15.0% and the tensile Index from 70 to 77 N m/g.

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Acknowledgments [39]

This work was carried out under the Academy of Finland’s Centres of Excellence Programme (2014–2019) and it was financially supported by the Finnish Bioeconomy Cluster (FiBiC LTD). Dr. Joseph Campbell (Aalto University) is acknowledged for the assistance in XPS measurements. Stora Enso is acknowledged for providing the SEM images. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

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