Development of new albumen based biocomposites formulations by injection moulding using chitosan as physicochemical modifier additive

Development of new albumen based biocomposites formulations by injection moulding using chitosan as physicochemical modifier additive J.E. Martín-Alfonsoa*, M. Félixb, A. Romerob, A. Guerrerob a Department of Chemical Engineering, University of Huelva, 21071 Huelva, Spain b Department of Chemical Engineering, University of Sevilla, 41012 Sevilla, Spain * Corresponding author. Tel.: +34 9599985; fax: +34 959219385 E-mail address: [email protected] (J.E. Martín Alfonso). 2 ABSTRACT In this study, albumen/chitosan (A/CH) based biocomposites were successfully prepared using injection moulding method. In particular, the influence of CH as physicochemical modifier additive in composite materials with A protein matrix in different concentrations in the range of 0–40 wt.% were considered. Biocomposites from A/CH were characterized by Fourier Transform Infrared Spectroscopy (FTIR), thermal analysis and Confocal Laser Scanning Microscopy (CLSM). Dynamic Mechanical Thermal Analysis (DMTA), tensile properties, water absorption capacity and standard tests to assess antimicrobial activities of the composites were also evaluated. FTIR analysis showed no new chemical bonds, suggesting that there was no chemical reaction and that the interaction was weak between the two biopolymers. DMTA spectra were qualitatively similar for A/CH biocomposites, and comparable to commercial low density polyethylene (LDPE). The incorporation of CH particles led to less stiff and less elastic composites. Thermal properties of albumen composite were affected by CH addition. Water absorption capacities of the modified composites were smaller than neat albumen composite. Keywords: A. Polymer-matrix composites (PMCs) B. Mechanical properties C. Thermal properties D. Injection moulding 3 1. Introduction Nowadays, there is a marked tendency to increase the use of “green materials” from renewable resources in order to avoid the impact that process technologies and products cause in the environment. This can be achieved by the synthesis of polymers from natural resources or by mixtures of biopolymers, such as polysaccharides and protein. The composites industry is especially involved in the maximization of these renewable materials [1, 2]. Egg albumen (A) is an excellent natural source of protein. A is a low cost material, compared with other proteins, and it is readily available as a by-product of dessert manufacturing industry. Moreover, A exhibits several interesting functionalities that make it attractive for composites industry, such as high transparency, an improved mechanical behavior and an easy processing by compression-moulding [3, 4]. However, in order to cover more demanding and specific purposes, enhanced and innovative characteristics are still required. In recent years, modification of albumen composites with polysaccharides has been a subject of great interest due to its direct impact on the final material properties [4]. Gonzalez-Gutierrez et al. [5] investigated the effect of starch (corn and potato) to a base formulation of albumen and glycerol and concluded that the resulting material properties strongly depend upon the selected procedure and its processing conditions. Optical and tensile properties were strongly affected by starch concentration. However, dynamic mechanical thermal properties proved to be insensitive to starch addition. In addition to starches, chitin and its derivatives are another interesting group of polysaccharides that could be used as additives or base ingredient for composites formulations. Chitosan (CH) is a natural polycationic polysaccharide derived from chitin by deacetylation in the presence of alkali. It has been investigated extensively for several decades for use in biosensors, artificial skin, bone substitutes, and water treatments [6]. Its importance resides in their biocompatibility, biodegradability, multiple functional groups, and solubility in aqueous medium [7]. Currently, CH is being used as a composite forming biopolymer which can have different mechanical, thermal, and antimicrobial properties [7-9]. The characteristics of chitosan composites depend on the structure achieved during its processing methods and the nature of its components, such as, the types and amounts of plasticizers, additives and/or copolymers used [10-12]. On the other hand, most studies have reported the influence of albumen and glycerol only on the properties of composites produced by thermo-plastic and thermo-mechanical processing method 4 [3-5], however it should be noticed that at our knowledge, there is not any paper that have studied the combination of A/CH to make biocomposites produced by inyection moulding. Injection moulding is a very versatile and commonly used method for producing three dimensional plastic goods. If the protein/polysaccharide-based materials could be processed with this technique, it would open up the possibility of introducing a wide range of protein/polysaccharide -based three dimensional goods onto the market for different applications. [13]. The objective of this work was to investigate the use of chitosan as physicochemical modifier additive to formulate a base formulation containing albumen and glycerol biocomposites. Thus, A/CH based biocomposites were prepared by injection moulding and morphologically, thermo-mechanically characterized. In addition to this, water absorption capacity and standard tests to assess antimicrobial activities of the composites were also evaluated. 2. Experimental 2.1. Materials Commercial spray-dried albumen (with 73 wt.% protein, 6wt.% ashes and 8 wt.% moisture) provided by OVOSEC S.A. (Spain) and chitosan (average molecular weigth = 2.29 105 g mol-1, 86.3% degree of deacetylation) supplied by Qingdao Fraken (China) were used as base materials for biocomposites manufacture. Glycerol (G), from Panreac Química, S.A. (Spain), was used as plasticizer. Additionally, and for the sake of comparison, commercial LDPE (Dow Chemical, Ref. 302R), with (Mw=2.07 105 g mol-1; Mw/Mn=5.8; ρ=0.924 g/cm3 and melt flow index 0.8 g/10 min (190ºC/2.16 kg) was used. 2.2. Preparation of albumen/chitosan based biocomposites Biocomposites were manufactured by a thermo-mechanical procedure which includes two stages, mixing of raw materials to obtain a dough-like mixture, and, then, injection moulding under pressure and heat. Ingredients (albumen, chitosan and glycerol) were mixed in a plastograph kneading tool Brabender Technologie K.G. (Germany). The mixing chamber could be considered as adiabatic, i.e. it was not heated or cooled. Mixing was carried out at 50 rpm for 10 min [3], using two counter rotating rollers turning with different angular velocities (ratio 3:2), and filled 75-90% of its full capacity. 5 A constant 40 wt.% plasticizer concentration was used, and five concentrations of chitosan (with respect to the total mass of mixture) were studied: 6, 10, 20, 30 and 40 wt.%. After the mixing process, the dough-like material was stored at -4ºC. Injection-moulded biocomposites were obtained, using a Thermo Scientific HAAKE MiniJet injection moulding machine. The nozzle and mould temperatures were 60ºC and 130ºC, respectively, and the injection pressure was 50 MPa, with a residence time in the mould of 60 s. Two types of moulds were used: one to obtain rectangular beams (50 x 10 x 3 mm3) for DMTA experiments, and the second one to obtain type IV probe defined by the Standard Test Method ASTM D638-99 [14] for Tensile Properties. 2.3 Characterization Fourier transform infrared (FTIR) spectra were obtained at room temperature using Digilab FTS3500ARX (Varian) apparatus, in a wavenumber range of 400–4000 cm−1 with a nominal resolution of 4 cm−1. The tensile properties were evaluated at room temperature using an Insight 10 kN Electromechanical Testing System (MTS, Eden Prairie, MN, USA). All tensile tests were stroke controlled with a constant cross head speed of 20 mm/min. According to the standard ASTM D638-99 [14], five tests were carried out for each sample. Dynamic Mechanical Thermal Analysis (DMTA) experiments were performed with a RSA3 (TA Instruments, New Castle, DE, USA), on rectangular probes using dual cantilever bending mode. Temperature sweeps were performed at a constant frequency (1 Hz) and strain within the linear viscoelastic region. The selected heating rate was 3ºC/min with a temperature range from -30 to 130. All tests were done in triplicate. Thermogravimetric analyses were carried out using Q50 DSC from TA Instruments (New Castle, USA). The samples were heated from room temperature to 600 ºC at a heating rate of 10ºC/min under a nitrogen atmosphere. The thermal properties of the samples were also determined with a Model Q100 DSC from TA Instruments (New Castle, USA). Accurately weighted (5-10 mg) samples sealed in hermetic aluminium pans. Heating rates of 10ºC/min, inside a temperature range of −30 to 180ºC, were applied. Water absorption measurements were based on standard ASTM D570-98 [15], using at least three 50mm x 10mm x 3 mm specimens immersed in distillate water for 24h at room temperature. The water uptake was calculated with Eq. (1), as follows: 𝑤𝑎𝑡𝑒𝑟 𝑢𝑝𝑡𝑎𝑘𝑒 (%)= [𝑊𝑡−𝑊𝑜 𝑊𝑜 ]𝑥100 (1) 6 where Wt and W0 are the weights of the specimen containing water and the dried specimen, respectively. Morphological modifications of the sample were evaluated through Confocal Laser Scanning Microscopy (CLSM, LSM 7 DUO) from Carl Zeiss Microscopy GmbH (Jena, Germany) was used to characterize the morphology of biocomposite materials. The CLSM was used in the fluorescent mode, and a wavelength of 405 nm from the laser was used. Wavelengths from 410 to 585 nm were analyzed. A 20× objective was used. It was not necessary to stain the sample due to the autofluorescent properties shown by the protein used. In vitro antibacterial behavior of the biocomposites was evaluated by inhibition zone experiments against Gram-negative bacterium (Escherichia coli). The composites were cut around 1x1 cm2 and then placed on nutrient agar plates coated with the bacteria with a concentration of approximately 105 CFU/ml. Finally, the plates were incubated at 37°C for 24 hours, and the inhibition zone was measured. At least, three repeats of each test were done and data shown have statistically significant values, i.e. they did not exceed a significance level of 0.05 in Student’s t-test and had a 95% confidence interval. 3. Results and discussion 3.1. FTIR spectroscopy FTIR spectra of A protein, CH and selected A/CH biocomposites are shown in Fig. 1. The relevant peaks in the spectrum of CH were similar to those described by other authors. The absorption peaks at 3440 cm-1 corresponding to the inter- and intra-molecular hydrogen bonding of both – NH2 and –OH stretching vibration, respectively [16]. Peaks around 1080 and 1370 cm-1 are due to saccharide structure and around 1637 cm-1 are due to carbonyl groups respectively [17]. The peak at 1655 cm-1 corresponding to N-acetyl groups of chitin, which indicates incomplete deacetylation [16]. The absorption band at 1370 cm-1 attributes the C–O stretching of primary alcoholic group (–CH2–OH). The absorption peak at 610 cm-1 indicates O–CN deformation [17]. On the other hand, A protein shows the peaks at 1500–1550 cm-1, 1600–1700 cm-1 and 3100–3500 cm- 1 correspond to the characteristic absorptions of amide II (N–H), amide I (C=O) and amide A, which are typical protein absorption bands [18]. Fig. 1b shows the FTIR spectra of A/CH biocomposites. Analysis of the relevant peaks indicated no new chemical bonds in the blends, suggesting that there was no chemical reaction between the two biopolymers, which is consistent with literature reports of albumen protein blends with other polysaccharides [4, 5]. However, the absorption 7 band at around 3440 cm−1 attributed to amino groups became broader and displaced to lower frequencies by about 2–10 cm-1 when CH content increase. These shifts may be attributed to other possible interactions include intermolecular hydrogen bonding between the anionic –COO– group of A protein and the cationic –NH3+ group in CH are capable of forming complexes with opposite charges. 3.2. Mechanical analysis 3.2.1 Tensile properties The tensile properties were tested by universal testing machine to determine the effect of CH content on tensile properties of modified biocomposites. Fig. 2 shows typical tensile stress–strain curves under a tension load at a constant speed. As shown in the figure, all the curves exhibited similar stress–strain behavior with an initial elastic segment followed by a plastic one and final fracture. Compared with pure A, biocomposites modified with CH exhibited higher stress at same strain. However, modified biocomposites showed more brittle tensile behavior. The failure strain of the A-based biocomposite was ~49% higher than that of the modified biocomposite with 20 wt.% CH. The tensile strength, determined from the stress–strain curve, decreased slightly with the chitosan content (Fig. 3). Additionally, the presence of CH caused a considerable decrease in the elongation at break (Fig. 3), A-based biocomposite exhibited elongation at break values that was ~78% better than those ascertained for modified biocomposite with 20 wt.% CH. These results suggest a low compatibility between A/CH biopolymers and the generation of heterogeneities in the protein matrix which decreased the overall cohesion forces in the matrix and acted as stress concentration points, inducing cracking make it more sensible to tensile test. 3.2.2. Dynamic mechanical thermal analysis The evolution of the storage modulus (E’) and loss tangent values (tan ) from -30 to 130ºC for selected A/CH biocomposites are shown in Fig. 4a-b, respectively. A-based biocomposite shows a continuous decay in the storage modulus although a more remarkable decay between 45ºC and 75ºC. This region is transitional between the glassy and the rubbery region where the side chain and main chain rotation becomes evident with increasing temperature. Regarding A/CH based biocomposites, the behaviour seen was never significantly altered, the storage modulus 8 was also smaller than that of the A-based biocomposite, and the presence of CH to protein matrix caused a shift in the E’ curves to smaller temperatures, indicating that CH increase the chain mobility. On the contrary, LDPE shows the greatest decay in E’ as temperature increases, with values of the storage modulus rapidly going down when temperature approaches polymer melting point. Moreover, the storage modulus values for A/CH based biocomposites were higher than those obtained with a commercial LDPE, over a temperature range studied. Fig. 4b shows the variation of tan  with temperature. The glass transition values (Tg) were determined from the peak of the loss tangent (ratio of loss modulus to storage modulus) [19]. The reduce area under the peak or damping denotes the stiffness in the composite. For A-based biocomposite, this peak is around 60ºC. As chitosan content is added the glass transition values move to lower temperatures, as a consequence of the increased the chain mobility due to the not strong attractive A/CH interactions. However, as the amount of CH increases the shift is expected to be greater but the particle size also increases in proportion, reducing the surface interaction and thus showing a relatively lower increment in Tg at a higher concentrations of CH. 3.3 Thermal characterization 3.3.1 TGA characterization Thermal decomposition of A/CH based biocomposites can be used to characterize biopolymer interactions. Fig. 5a exhibits the TG curves for A protein and CH. For both biopolymers, it can be observed that weight loss took place in two stages. The first one starts about 35ºC. This stage is assigned to the loss of water. For CH, the second stage starts at 250ºC and reaches a maximum at 298ºC with a weight loss of 55.2%. This stage corresponds to the decomposition (thermal and oxidative), vaporization and elimination of volatile products [20]. For pure A protein the total degradation was completed at 425ºC. Regarding biocomposites materials, the weight loss rate curves (DTG), show at least three thermal events, labelled as peaks P1 to P3 (Fig. 5b). Among them, peaks P2 and P3 (located at 250 and 310ºC) are the most relevant ones and strongly affected by biocomposite composition. The first (P1) peak (located close to 175ºC) of the thermal degradation of composite may be associated to the loss of free and bonded water respectively, whereas the fourth (P3) peak (close 310ºC) would be related to the proteins volatilization [21]. The addition of chitosan provokes that the peaks P2 and P3 are overlapped. Likewise, according to data shown 9 in Fig. 5a, glycerol volatilization start close 100ºC and progressively increase up to the highest weight loss rate close to 237ºC. This result suggests occurrence of peak P2 is mainly related to the glycerol used as composite plasticiser. Similar results were founded by Chen et al. [22] for soy-protein-based materials. Table 1 shows more relevant parameters obtained from the thermogravimetric analysis: The temperature for the onset of thermal decomposition (Tonset), the temperature at which decomposition rate is maximum (Tmax) and the percentage of non-degraded residue. As can be deduced from Table, the higher the CH content is, the Tonset appears before. Hence, thermal stability of A/CH biocomposite increases by decreasing CH content. All the composites studied show very similar percentage of non-degraded residue (w= 17.08±1.34), not significantly affected by the inclusion of CH. 3.3.2 DSC characterization Fig. 6 presents differential scanning calorimetry (DSC) thermograms of A/CH based biocomposites. As seen in Fig. 6, the DSC spectra exhibit one main thermal event that occurs in the medium temperature region (40-80ºC), and corresponds to the glass transition temperature (Tg) of A/CH in samples, and appeared in all the biocomposites samples. The Tg of A/CH based biocomposites decrease with the increase in chitosan contents. The addition of CH reduced the intermolecular forces between biopolymer chains and consequently the overall cohesion and thereby the Tg to less temperature. These observations are in accordance with the mechanical properties previously discussed. Thermal behavior influences how biopolymers are dispersed and, therefore, composite morphology. Fig. 7 shows CLSM observations for a selected A/CH biocomposites. This figure highlights a difference in the organization of the systems according to CH presence in biocomposites. For the system without CH, homogeneities in the distribution of A protein were evidenced by the presence of dark regions corresponding to glycerol domains, embedded in a continuous fluorescent phase of homogeneous A protein. When CH was present in the sample, a major change in the texture of the image was observed, with an inhomogeneous distribution of the fluorescence which can be related to the formation of CH crosslink within the A matrix. From these overall CLSM observations, it appears that, whether albumen protein or chitosan, the two components were distinguished with a relatively different distribution. These observations provide evidence that in the composites, A/CH were localized in different phases. 16 Figure Captions Fig. 1. FTIR spectra of: (a) pure albumen (A) and chitosan (CH) biopolymers and (b) selected A/CH biocomposites samples. Fig. 2. Stress-strain curves of A/CH biocomposites as a function of the chitosan contents. Fig. 3. The effect of chitosan addition on Young modulus, tensile strength and elongation at break of the biocomposites. Fig. 4. Temperature dependence of storage modulus (E’) and loss tangent (tan for selected A/CH biocomposites samples. Fig. 5. TGA curves for (a) pure albumen (A) and chitosan (CH) biopolymers and (b) some of their corresponding biocomposites samples. Weight loss vs. temperature plot and its derivative function. Fig. 6. DSC curves of selected A/CH biocomposites samples. A: Albumen, CH: chitosan. Fig. 7. CLSM micrographs of selected biocomposites: a) A/CH: 100/0; b) A/CH: 80/20; c) A/CH: 70/30; d) A/CH: 60/40. A: Albumen, CH: chitosan. Fig. 8. Water uptake capacity, after 24 h, for A/CH biocomposites with chitosan contents. Fig. 9. Photograph showing growth (E. coli) on surface of selected biocomposites: a) A/CH: 100/0; b) A/CH: 90/10; c) A/CH: 80/20; d) A/CH: 60/40. A: Albumen, CH: chitosan. 17 4500 4000 3500 3000 2500 2000 1500 1000 500 Amide II A Transmittance CH a) Amide I b) A/CH:90/10 A/CH:94/6 A/CH:70/30 A/CH:60/40 Transmittance Wavenumber (cm-1) A/CH:100/0 Figure 1 18 010 20 30 40 50 60 0 1 2 40% CH 30% CH 10% CH 20% CH 6% CH Stress (Mpa) Strain (%) 0% CH Figure 2 19 0 2 4 6 8 Tensile Strength Elongation at Break ( x0.1) Young's Module Elongation at Break (%) x0.1 Tensile Strength (MPa) 0 20 40 60 40% CH 30% CH 20% CH 10% CH 6% CH Young's Modulus (MPa) 0% CH Figure 3 20 -40 -20 0 20 40 60 80 100 120 140 0,0 0,1 0,2 0,3 1,0 1,2 1E8 1E9 1E10 A/CH:100/0 A/CH:80/20 A/CH:60/40 LDPE b) tan  Temperature (ºC) A/CH:100/0 A/CH:80/20 A/CH:60/40 LDPE E' (Pa) a) Figure 4 21 0 20 40 60 80 100 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0100 200 300 400 500 600 0 20 40 60 80 100 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Albumen (A) Chitosan (CH) Deriv. weight (%/ºC) Deriv. weight (%/ºC) Weight (%) a) b)A/CH: 100/0 A/CH: 70/30 A/CH: 60/40 P3 P2 Weight (%) Temperature (ºC) P1 Figure 5 22 -50 0 50 100 150 200 A/CH:80/20 A/CH:90/10 Tg (54.2ºC) Tg (57.5ºC) Heat flow (W/g) Temperature (ºC) Tg (78.8ºC) A/CH:100/0 0.1 W/g Figure 6 23 Figure 7 A B C D 24 010 20 30 40 0 5 10 15 20 25 30 35 % Water uptake-24 h CH content % 6 Reference Figure 8 25 Figure 9 B A D C