- Email: [email protected]
Effect of crystalline substances in biodegradable films
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
Contents lists available at ScienceDirect
Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd
Effect of crystalline substances in biodegradable films Patrick Frohberg a , Markus Pietzsch b , Joachim Ulrich a,∗ a
Martin-Luther-Universität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik/TVT, D-06099 Halle, Germany Martin-Luther-Universität Halle-Wittenberg, Faculty of Natural Sciences I, Institute of Pharmacy, Department of Downstream Processing, D-06099 Halle, Germany
b
a b s t r a c t Films made of sodium caseinate and gelatine were prepared by casting method from a water solution containing glycerol as a plasticizer to obtain environmentally friendly and fully biodegradable materials for agricultural and packaging applications. Additionally, enzymatic protein cross-linking by microbial transglutaminase was applied. Potassium nitrate (KNO3 ) was used as additive to investigate the influence of crystallization on the physical properties of protein films. Mechanical properties (tensile strength and elongation at break) of the protein films were determined versus ratio of protein to potassium nitrate in the presence and absence of microbial transglutaminase. Furthermore, optical properties (film formation and consistency, surface texture, crystal size, shape, and distribution of incorporated potassium nitrate) were examined. An increase of 122% and 177% in the elongation of the films was adopted due to the crystallization of KNO3 in enzymatic-modified sodium caseinate and gelatine films, respectively. Pure sodium caseinate films distinguished ultimate tensile strengths of 4.95 MPa, while MTG-treated gelatine films achieved ultimate tensile strengths of 13.52 MPa. Altogether, the most appropriate overall mechanical performance was obtained for KNO3 /protein ratios of 1:6 in enzymatic-modified films. Furthermore, an increasing content of crystalline KNO3 results in increasing thickness, rough surfaces, decreased opacity, and whitish coloring of the films. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Crystallization; Biodegradable films; Sodium caseinate; Gelatine; Potassium nitrate
1.
Introduction
Today, the development of innovative materials to substitute synthetic polymers has become an important challenge. Among these materials are biopolymers from vegetable or animal proteins (Arvanitoyannis, 2006). These materials offer a unique set of properties that make them favorable for applications chiefly in market sectors such as packaging, agriculture, electronics, automotive, and medicine. Derived from natural sources, these biodegradable, biocompatible and non-toxic polymers meet the growing demands for environment-friendly products (Clarinval and Halleux, 2005). In this study, biodegradable films and composites for agricultural applications (greenhouse, walk-in tunnel, low tunnel covers, and mulching) are in the focus. Petroleum-
∗
based plastics often remain undegraded after discard and a time-consuming and uneconomical recycling is unavoidable. The adoption of biopolymers avoids the removal of residual materials from the growing environment after functional compliance (Espi et al., 2006; Joo et al., 2005). Furthermore, degradation could offer additional benefit by controlling the release of incorporated agrochemicals, fertilizers and pesticides (Clarinval and Halleux, 2005). The film forming and thermoplastic properties due to their abilities to form weak intermolecular interactions, i.e. hydrogen, electrostatic and hydrophobic bonds make, i.e. sodium caseinate a promising raw material for the production of biodegradable films and coatings (Audic and Chaufer, 2005). However, for the majority of the mentioned applications, the improvement of the physicochemical properties
Corresponding author. Tel.: +49 3455528400; fax: +49 3455527358. E-mail address: [email protected] (J. Ulrich). Received 12 December 2008; Received in revised form 5 October 2009; Accepted 27 January 2010 0263-8762/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cherd.2010.01.037
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
of protein-based materials is of first importance. Therefore, chemical modifications of biopolymers and the development of specific additives (cross-linking agents and plasticizers) are already well investigated (Clarinval and Halleux, 2005). Furthermore, an enzymatic catalyzed cross-linking of proteins associated with a controlled additive crystallization appears to be a promising way to improve physicochemical properties of biodegradable films and coatings. While the effects of transglutaminase treatment on the film properties of various proteins have been extensively studied the crystallization of selective additives in protein-based materials is a completely new approach for the optimization of the materials (Flanagan et al., 2003; Lorenzen, 2000; Oh et al., 2004; Tang et al., 2005; Wang et al., 2007; Yi et al., 2006). Therefore, the objective of this study is to determine the influence of crystallized potassium nitrate on the mechanical and optical properties of sodium caseinate and gelatine films in the presence and absence of microbial transglutaminase.
2.
Methods and materials
2.1.
Materials
Sodium caseinate (NaCas) (protein 88%, fat 1.5%, lactose 0.2%, ash 4.5%, and water 6%) was provided by BMI e.G. (Landshut, Germany). Gelatine (high grade, type 280 bloom) was supplied by Gelita Europe (Eberbach, Germany). Microbial transglutaminase (MTG) (ACTIVA® WM, nominal activity = 100 U/g of powder) was purchased from Ajinomoto Europe Sales GmbH (Hamburg, Germany). Analytical grade glycerol and potassium nitrate were supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany).
2.2.
100 mm using a double-bladed roller knife. Film thickness was determined from the mean of three measurements across each specimen using a micrometer. Subsequently, the sample films were tested with a grip separation of 100 mm and a crosshead speed of 50 mm/min.
2.4.
Film morphology
Optical properties (film surface, opacity, coloring, crystal size, shape, and distribution) were investigated using digital photography and light microscopy. Film samples were stored in a desiccator for at least 48 h to assure constant observation conditions. Micrographs were examined at magnifications of 5–335×.
2.5.
Statistical analysis
Analysis of variance (ANOVA; Statistica® 8, StatSoft, Tulsa, OK, USA) was used to indicate a significant difference (P < 0.05) amongst the means. Five replications were employed for each sample.
3.
Results and discussion
3.1.
Film formation
Flexible, transparent, and light yellowish films with homogenous textures were obtained after drying the film-forming solutions containing NaCas and glycerol. Films made of gelatin showed transparent, homogenous, and non-sticky overall properties. The crystallization of the potassium nitrate due to the evaporation of the solvent results in increased film thickness, rough surfaces and increasing opacity.
Film formation and preparation 3.2.
Protein films were prepared by casting. An aqueous solution of sodium caseinate or gelatine (5%, w/w), glycerol (2.5%, w/w), and potassium nitrate (KNO3 ) in different concentrations (KNO3 /protein ratios of 1:12 to 1:1.33) was magnetically stirred for 30 min at 90 ◦ C in order to get a homogenous solution. For the addition of microbial transglutaminase (25 U/g NaCas) the solution was cooled to room temperature, the pH was set to 7 by adding 1 M sodium hydroxide (NaOH), and warmed up to 50 ◦ C. To prevent air entrapment in the films the solution was centrifuged for 1 min at 177 × g and 50 ◦ C before the casting process. The film forming solution was spread onto a polytetrafluoroethylene (PTFE) plate and the film, dried for 48 h at room temperature and peeled off after the water evaporated. In order to provide homogeneous films in terms of thickness und surface conditions millimeter accurate screws were used to adjust the PTFE plates and to assure an equal wettability. To ensure constant relative humidity (50 ± 2% RH) conditions the films were conditioned in a closed tank containing saturated solutions of Ca(NO3 )2 ·4H2 O for at least 48 h at room temperature.
2.3.
1149
Tensile strength
Tensile strength values of sodium caseinate in dependence of additive concentration and enzyme catalyzed cross-linking of the protein are given in Fig. 1. The results for NaCas-films show a distinct dependence of the potassium nitrate concentration. The highest tensile strength of 4.95 MPa was achieved with pure NaCas-films
Mechanical properties
Mechanical properties (tensile strength and elongation) were measured according to the standard procedure of DIN EN ISO 527-3 using a material testing machine (BDO-FB0.5TH, Zwick/Roell, Ulm, Germany). Therefore the films were cut into test stripes with a wide of 15 mm and a length of at least
Fig. 1 – Tensile strength of MTG-treated (+MTG) and non-treated (−MTG) NaCas-films at different KNO3 concentrations.
1150
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
Fig. 2 – Tensile strength of MTG-treated (+MTG) and non-treated (−MTG) gelatine films at different KNO3 concentrations.
Fig. 3 – Elongation at break of MTG-treated (+MTG) and non-treated (−MTG) NaCas-films at different KNO3 concentrations.
without addition of KNO3 and MTG. With increasing additive concentration the tensile strength of the films were decreasing autonomous of an enzymatic cross-linking via MTG to 1.64 and 1.28 MPa, respectively. Generally, an enzymatic modification of NaCas-films exhibits minor changes within the standard deviation except for a KNO3 /NaCas ratio of 1:4. In this case, the cross-linking effect yields a tensile strength increase of 57%. As shown in Fig. 2, non-crystalline films (KNO3 :protein 0:1) made of gelatine showed significantly higher tensile strength values (P < 0.05) than NaCas-films independent of an enzymatic modification via MTG. The enzymatic cross-linking of pure gelatine films achieved the largest effect by increasing the tensile strength from 9.41 to 13.52 MPa. A marked decrease of film tensile strength depending on the incorporated KNO3 crystals was determined by analyzing the gelatine films prepared both in the presence and absence of MTG. Gelatine films containing crystallized potassium nitrate in a relation between 1:12 and 1:4 in respect of gelatine showed proximate tensile strength in the range of 4–6 MPa. A further enhancement of the KNO3 fraction slightly decreased tensile strength to 3.64 and 2.96 MPa, respectively. Altogether, gelatine films revealed higher tensile strength values compared to protein films made of sodium caseinate. This behavior appears to be related to the protein chain structure of the current protein. NaCas is generally classified as unordered protein containing few ␣-helical and -structures (Siew et al., 1999). Consequently, films made of NaCas exhibit a less organized structure compared to gelatine films, which results in a reduced molecular packaging strength (Chambi and Grosso, 2006).
enhancement up to a ratio of 1:1.33. An increase of 122% was adopted by incorporating KNO3 to enzymatic-modified samples with 1:6 ratios. At KNO3 /NaCas ratios above 1:2 a considerable deterioration of films mechanical performance was obtained. This circumstance is preferential due to the formation of very large crystalline structures that damaged the macromolecular textures (predominately in terms of surface irregularities) of the films. On this weak spots the films were preferable disrupted during the measurement procedure. In addition, the elongation of crystallized gelatine films in dependence of an enzymatic modification has been investigated (Fig. 4). In comparison to pure gelatine films a crystallization of potassium nitrate results a considerable increase of film elongations. In particularly, a KNO3 :gelatine ratio of 1:2 improved the elongation of MTG-treated films by 177%. Only minor differences in films elongations between ratios of 1:6 and 1:1.33 were obtained. Generally, films made of sodium caseinate featured higher elongations (up to 201%) compared to gelatine with max-
3.3.
Elongation
Furthermore, the effect of crystallized potassium nitrate and the additional influence of an enzymatic protein cross-linking on the elongation of the protein films was determined. As shown in Fig. 3, MTG-treated NaCas-films achieved significant higher elongations (P < 0.05) compared to non-treated films with the exception of samples without crystallized KNO3 . Films made of NaCas show an increase of the elongation with increasing potassium nitrate concentrations until a ratio of 1:6 followed by a considerable decrease with further KNO3
Fig. 4 – Elongation at break of MTG-treated (+MTG) and non-treated (−MTG) gelatine films at different KNO3 concentrations.
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
1151
Fig. 5 – Light microscopy exposures of incorporated potassium nitrate crystals at KNO3 /NaCas ratios of (a) 1:6, (b) 1:4, and (c) 1:1.33; −MTG. imum values of 147%. Contrariwise, gelatine films were offering conspicuous higher tensile strengths to the point of 13.52 MPa in contrast to NaCas-films with maximal values of 4.95 MPa. In general, enzymatic cross-linked NaCas and gelatine films with KNO3 /protein ratios of 1:6 offer the most applicable combination in reference to the total mechanical
performance. The mechanical properties of protein-based films and coatings are chiefly associated with intermolecular and intramolecular interactions inside the protein network depending on arrangement and orientation of the polymer chains (Chambi and Grosso, 2006). The alteration of films mechanical characteristics due to incorporated crystals appears to be related to a weakening of the intermolecular
Fig. 6 – Digital photography exposures of protein films at various potassium nitrate concentrations: (a) 1:4, NaCas; (b) 1:2, NaCas; (c) 1:2, gelatine; (d) 1:1.33, NaCas; −MTG.
1152
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
bonds. This results in increasing elongations and decreasing tensile strengths.
3.4.
Morphology
Depending on the potassium nitrate concentration miscellaneous crystal structures and sizes were obtained. As presented in Fig. 5, independent of an enzymatic cross-linking the typical orthorhombic (aragonite-type) structure was observed with KNO3 /NaCas ratios of 1:6 and 1:4 at room temperature. Average crystal sizes between 30 and 200 m were observed at the mentioned concentrations illustrated in Fig. 5(a) and (b). With increasing supersaturation due to the increase of the KNO3 concentration the crystal size increased clearly in conjunction with a structural alternation to a more complicated crystallographic form shown in Fig. 5(c). Brittle columnar sprawling crystals with an effective length up to 100 mm with partial fractures were observed at ratios of 1:1.33. Mentionable differences referring to crystal sizes and structures of NaCas and gelatine films (enzymatically treated and non-treated) could not be ascertained. Protein films with crystallized potassium nitrate at ratios less than 1:6 showed no macroscopic visible crystals in the structure. In these experiments, transparent and smooth structured films were observed. The exposures in Fig. 6(a) show crystalline agglomerates in most instances coexisting with adjacent areas of uniform distributed potassium nitrate crystals in the case of 1:4 ratios. Homogeneously distributed columnar crystals with sizes up to 10 mm were obtained by increasing the KNO3 concentration to ratios of 1:2 (Fig. 6(b)) in NaCas-films. Films made of gelatine show crystalline agglomerates on the surface of the films at ratios of 1:2 (Fig. 6(c)). A further increase of the potassium nitrate concentration (1:1.33, Fig. 6(d)) tends to result in a macroscopic acicular crystalline network after solvent evaporation. The crystals pervaded the entire protein film and protruded at both sides. Under these circumstances the incorporated crystals affect dramatically the overall mechanical performance to the negative. Deviant morphological characteristics of cross-linked films and of samples without enzymatic modification were not ascertained.
4.
Conclusions
This study clearly showed the potential of an inner-film crystallized additive to enhance the mechanical properties of protein-based biodegradable films and coatings as the general basis to expand the application to new fields. Especially, the crystallization of potassium nitrate induced a considerable increase of 122% in the elongation of NaCas-films associated with a minor decrease of film tensile strength. Actually, an improvement of films elongation up to 177% was achieved for crystallized and enzymatic cross-linked gelatine films. However, the great increase of film elongation was accompanied with an explicit decrease of the tensile strength in the case of gelatine. The synergistic effect of potassium nitrate crystallization and enzymatic cross-linking of the protein offered the highest potential in terms of enhancing the overall mechanical performance. Precisely, at KNO3 /NaCas ratios of 1:4 the introduction of cross-linkages increased films tensile strength of 57% and elongation of 137%, respectively. Besides the efforts to improve the physical properties of biodegradable films and coatings additional benefits are able
to facilitate a market share increase or the development of new markets, respectively. Therefore, the selection of reasonable crystallizing additives, e.g. potassium nitrate due to its applications as agricultural fertilizer or for food preservation are promising ways to establish biodegradable films in industry and society.
Acknowledgement The authors want to acknowledge the financial support of the Fachagentur Nachwachsende Rohstoffe e.V. (FNR) for the work presented here.
References Arvanitoyannis, I.S., 2006, Biodegradable edible films and coatings based on protein resources: physical properties and applications in food quality management, In Mallapragada, S.K. and Narasim, B., Narasim, B. (Eds.), Handbook of Biodegradable Polymeric Materials and their Applications (American Scientific Publishers, Stevenson Ranch, USA), pp. 200–235. (American Scientific Publishers, Stevenson Ranch, USA). Audic, J.-L. and Chaufer, B., 2005, Influence of plasticizers and crosslinking on the properties of biodegradable films made from sodium caseinate. European Polymer Journal, 41(8): 1934–1942. Chambi, H. and Grosso, C., 2006, Edible films produced with gelatin and casein cross-linked with transglutaminase. Food Research International, 39(4): 458–466. Clarinval, A.-M. and Halleux, J., 2005, Classification of biodegradable polymers, in Biodegradable Polymers for Industrial Applications, Smith, R. (ed) (Woodhead Publishing Limited, Cambridge, England), pp. 3–29. (Woodhead Publishing Limited, Cambridge, England). Espi, E., Salmeron, A., Fontecha, A., Garcia, Y. and Real, A.I., 2006, Plastic films for agricultural applications. Journal of Plastic Film & Sheeting, 22(2): 85–102. Flanagan, J., Gunning, Y. and FitzGerald, R.J., 2003, Effect of cross-linking with transglutaminase on the heat stability and some functional characteristics of sodium caseinate. Food Research International, 36(3): 267–274. Joo, S.B., Kim, M.N., Im, S.S. and Yoon, J.S., 2005, Biodegradation of plastics in compost prepared at different composting conditions. Macromolecular Symposia, 224: 355–365. Lorenzen, P.C., 2000, Techno-functional properties of transglutaminase-treated milk proteins. Milchwissenschaft-Milk Science International, 55(12): 667–670. Oh, J.H., Wang, B., Field, P.D. and Aglan, H.A., 2004, Characteristics of edible films made from dairy proteins and zein hydrolysate cross-linked with transglutaminase. International Journal of Food Science and Technology, 39(3): 287–294. Siew, D.C.W., Heilmann, C., Easteal, A.J. and Cooney, R.P., 1999, Solution and film properties of sodium caseinate/glycerol and sodium caseinate/polyethylene glycol edible coating systems. Journal of Agricultural and Food Chemistry, 47(8): 3432–3440. Tang, C., Yang, X.Q., Chen, Z., Wu, H. and Peng, Z.Y., 2005, Physicochemical and structural characteristics of sodium caseinate biopolymers induced by microbial transglutaminase. Journal of Food Biochemistry, 29(4): 402–421. Wang, J.S., Zhao, M.M., Yang, X.Q., Jiang, Y.M. and Chun, C., 2007, Gelation behavior of wheat gluten by heat treatment followed by transglutaminase cross-linking reaction. Food Hydrocolloids, 21(2): 174–179. Yi, J.B., Kim, Y.T., Bae, H.J., Whiteside, W.S. and Park, H.J., 2006, Influence of transglutaminase-induced cross-linking on properties of fish gelatin films. Journal of Food Science, 71(9): E376–E383.
Contents lists available at ScienceDirect
Chemical Engineering Research and Design journal homepage: www.elsevier.com/locate/cherd
Effect of crystalline substances in biodegradable films Patrick Frohberg a , Markus Pietzsch b , Joachim Ulrich a,∗ a
Martin-Luther-Universität Halle-Wittenberg, Zentrum für Ingenieurwissenschaften, Verfahrenstechnik/TVT, D-06099 Halle, Germany Martin-Luther-Universität Halle-Wittenberg, Faculty of Natural Sciences I, Institute of Pharmacy, Department of Downstream Processing, D-06099 Halle, Germany
b
a b s t r a c t Films made of sodium caseinate and gelatine were prepared by casting method from a water solution containing glycerol as a plasticizer to obtain environmentally friendly and fully biodegradable materials for agricultural and packaging applications. Additionally, enzymatic protein cross-linking by microbial transglutaminase was applied. Potassium nitrate (KNO3 ) was used as additive to investigate the influence of crystallization on the physical properties of protein films. Mechanical properties (tensile strength and elongation at break) of the protein films were determined versus ratio of protein to potassium nitrate in the presence and absence of microbial transglutaminase. Furthermore, optical properties (film formation and consistency, surface texture, crystal size, shape, and distribution of incorporated potassium nitrate) were examined. An increase of 122% and 177% in the elongation of the films was adopted due to the crystallization of KNO3 in enzymatic-modified sodium caseinate and gelatine films, respectively. Pure sodium caseinate films distinguished ultimate tensile strengths of 4.95 MPa, while MTG-treated gelatine films achieved ultimate tensile strengths of 13.52 MPa. Altogether, the most appropriate overall mechanical performance was obtained for KNO3 /protein ratios of 1:6 in enzymatic-modified films. Furthermore, an increasing content of crystalline KNO3 results in increasing thickness, rough surfaces, decreased opacity, and whitish coloring of the films. © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Crystallization; Biodegradable films; Sodium caseinate; Gelatine; Potassium nitrate
1.
Introduction
Today, the development of innovative materials to substitute synthetic polymers has become an important challenge. Among these materials are biopolymers from vegetable or animal proteins (Arvanitoyannis, 2006). These materials offer a unique set of properties that make them favorable for applications chiefly in market sectors such as packaging, agriculture, electronics, automotive, and medicine. Derived from natural sources, these biodegradable, biocompatible and non-toxic polymers meet the growing demands for environment-friendly products (Clarinval and Halleux, 2005). In this study, biodegradable films and composites for agricultural applications (greenhouse, walk-in tunnel, low tunnel covers, and mulching) are in the focus. Petroleum-
∗
based plastics often remain undegraded after discard and a time-consuming and uneconomical recycling is unavoidable. The adoption of biopolymers avoids the removal of residual materials from the growing environment after functional compliance (Espi et al., 2006; Joo et al., 2005). Furthermore, degradation could offer additional benefit by controlling the release of incorporated agrochemicals, fertilizers and pesticides (Clarinval and Halleux, 2005). The film forming and thermoplastic properties due to their abilities to form weak intermolecular interactions, i.e. hydrogen, electrostatic and hydrophobic bonds make, i.e. sodium caseinate a promising raw material for the production of biodegradable films and coatings (Audic and Chaufer, 2005). However, for the majority of the mentioned applications, the improvement of the physicochemical properties
Corresponding author. Tel.: +49 3455528400; fax: +49 3455527358. E-mail address: [email protected] (J. Ulrich). Received 12 December 2008; Received in revised form 5 October 2009; Accepted 27 January 2010 0263-8762/$ – see front matter © 2010 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cherd.2010.01.037
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
of protein-based materials is of first importance. Therefore, chemical modifications of biopolymers and the development of specific additives (cross-linking agents and plasticizers) are already well investigated (Clarinval and Halleux, 2005). Furthermore, an enzymatic catalyzed cross-linking of proteins associated with a controlled additive crystallization appears to be a promising way to improve physicochemical properties of biodegradable films and coatings. While the effects of transglutaminase treatment on the film properties of various proteins have been extensively studied the crystallization of selective additives in protein-based materials is a completely new approach for the optimization of the materials (Flanagan et al., 2003; Lorenzen, 2000; Oh et al., 2004; Tang et al., 2005; Wang et al., 2007; Yi et al., 2006). Therefore, the objective of this study is to determine the influence of crystallized potassium nitrate on the mechanical and optical properties of sodium caseinate and gelatine films in the presence and absence of microbial transglutaminase.
2.
Methods and materials
2.1.
Materials
Sodium caseinate (NaCas) (protein 88%, fat 1.5%, lactose 0.2%, ash 4.5%, and water 6%) was provided by BMI e.G. (Landshut, Germany). Gelatine (high grade, type 280 bloom) was supplied by Gelita Europe (Eberbach, Germany). Microbial transglutaminase (MTG) (ACTIVA® WM, nominal activity = 100 U/g of powder) was purchased from Ajinomoto Europe Sales GmbH (Hamburg, Germany). Analytical grade glycerol and potassium nitrate were supplied by Carl Roth GmbH & Co. KG (Karlsruhe, Germany).
2.2.
100 mm using a double-bladed roller knife. Film thickness was determined from the mean of three measurements across each specimen using a micrometer. Subsequently, the sample films were tested with a grip separation of 100 mm and a crosshead speed of 50 mm/min.
2.4.
Film morphology
Optical properties (film surface, opacity, coloring, crystal size, shape, and distribution) were investigated using digital photography and light microscopy. Film samples were stored in a desiccator for at least 48 h to assure constant observation conditions. Micrographs were examined at magnifications of 5–335×.
2.5.
Statistical analysis
Analysis of variance (ANOVA; Statistica® 8, StatSoft, Tulsa, OK, USA) was used to indicate a significant difference (P < 0.05) amongst the means. Five replications were employed for each sample.
3.
Results and discussion
3.1.
Film formation
Flexible, transparent, and light yellowish films with homogenous textures were obtained after drying the film-forming solutions containing NaCas and glycerol. Films made of gelatin showed transparent, homogenous, and non-sticky overall properties. The crystallization of the potassium nitrate due to the evaporation of the solvent results in increased film thickness, rough surfaces and increasing opacity.
Film formation and preparation 3.2.
Protein films were prepared by casting. An aqueous solution of sodium caseinate or gelatine (5%, w/w), glycerol (2.5%, w/w), and potassium nitrate (KNO3 ) in different concentrations (KNO3 /protein ratios of 1:12 to 1:1.33) was magnetically stirred for 30 min at 90 ◦ C in order to get a homogenous solution. For the addition of microbial transglutaminase (25 U/g NaCas) the solution was cooled to room temperature, the pH was set to 7 by adding 1 M sodium hydroxide (NaOH), and warmed up to 50 ◦ C. To prevent air entrapment in the films the solution was centrifuged for 1 min at 177 × g and 50 ◦ C before the casting process. The film forming solution was spread onto a polytetrafluoroethylene (PTFE) plate and the film, dried for 48 h at room temperature and peeled off after the water evaporated. In order to provide homogeneous films in terms of thickness und surface conditions millimeter accurate screws were used to adjust the PTFE plates and to assure an equal wettability. To ensure constant relative humidity (50 ± 2% RH) conditions the films were conditioned in a closed tank containing saturated solutions of Ca(NO3 )2 ·4H2 O for at least 48 h at room temperature.
2.3.
1149
Tensile strength
Tensile strength values of sodium caseinate in dependence of additive concentration and enzyme catalyzed cross-linking of the protein are given in Fig. 1. The results for NaCas-films show a distinct dependence of the potassium nitrate concentration. The highest tensile strength of 4.95 MPa was achieved with pure NaCas-films
Mechanical properties
Mechanical properties (tensile strength and elongation) were measured according to the standard procedure of DIN EN ISO 527-3 using a material testing machine (BDO-FB0.5TH, Zwick/Roell, Ulm, Germany). Therefore the films were cut into test stripes with a wide of 15 mm and a length of at least
Fig. 1 – Tensile strength of MTG-treated (+MTG) and non-treated (−MTG) NaCas-films at different KNO3 concentrations.
1150
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
Fig. 2 – Tensile strength of MTG-treated (+MTG) and non-treated (−MTG) gelatine films at different KNO3 concentrations.
Fig. 3 – Elongation at break of MTG-treated (+MTG) and non-treated (−MTG) NaCas-films at different KNO3 concentrations.
without addition of KNO3 and MTG. With increasing additive concentration the tensile strength of the films were decreasing autonomous of an enzymatic cross-linking via MTG to 1.64 and 1.28 MPa, respectively. Generally, an enzymatic modification of NaCas-films exhibits minor changes within the standard deviation except for a KNO3 /NaCas ratio of 1:4. In this case, the cross-linking effect yields a tensile strength increase of 57%. As shown in Fig. 2, non-crystalline films (KNO3 :protein 0:1) made of gelatine showed significantly higher tensile strength values (P < 0.05) than NaCas-films independent of an enzymatic modification via MTG. The enzymatic cross-linking of pure gelatine films achieved the largest effect by increasing the tensile strength from 9.41 to 13.52 MPa. A marked decrease of film tensile strength depending on the incorporated KNO3 crystals was determined by analyzing the gelatine films prepared both in the presence and absence of MTG. Gelatine films containing crystallized potassium nitrate in a relation between 1:12 and 1:4 in respect of gelatine showed proximate tensile strength in the range of 4–6 MPa. A further enhancement of the KNO3 fraction slightly decreased tensile strength to 3.64 and 2.96 MPa, respectively. Altogether, gelatine films revealed higher tensile strength values compared to protein films made of sodium caseinate. This behavior appears to be related to the protein chain structure of the current protein. NaCas is generally classified as unordered protein containing few ␣-helical and -structures (Siew et al., 1999). Consequently, films made of NaCas exhibit a less organized structure compared to gelatine films, which results in a reduced molecular packaging strength (Chambi and Grosso, 2006).
enhancement up to a ratio of 1:1.33. An increase of 122% was adopted by incorporating KNO3 to enzymatic-modified samples with 1:6 ratios. At KNO3 /NaCas ratios above 1:2 a considerable deterioration of films mechanical performance was obtained. This circumstance is preferential due to the formation of very large crystalline structures that damaged the macromolecular textures (predominately in terms of surface irregularities) of the films. On this weak spots the films were preferable disrupted during the measurement procedure. In addition, the elongation of crystallized gelatine films in dependence of an enzymatic modification has been investigated (Fig. 4). In comparison to pure gelatine films a crystallization of potassium nitrate results a considerable increase of film elongations. In particularly, a KNO3 :gelatine ratio of 1:2 improved the elongation of MTG-treated films by 177%. Only minor differences in films elongations between ratios of 1:6 and 1:1.33 were obtained. Generally, films made of sodium caseinate featured higher elongations (up to 201%) compared to gelatine with max-
3.3.
Elongation
Furthermore, the effect of crystallized potassium nitrate and the additional influence of an enzymatic protein cross-linking on the elongation of the protein films was determined. As shown in Fig. 3, MTG-treated NaCas-films achieved significant higher elongations (P < 0.05) compared to non-treated films with the exception of samples without crystallized KNO3 . Films made of NaCas show an increase of the elongation with increasing potassium nitrate concentrations until a ratio of 1:6 followed by a considerable decrease with further KNO3
Fig. 4 – Elongation at break of MTG-treated (+MTG) and non-treated (−MTG) gelatine films at different KNO3 concentrations.
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
1151
Fig. 5 – Light microscopy exposures of incorporated potassium nitrate crystals at KNO3 /NaCas ratios of (a) 1:6, (b) 1:4, and (c) 1:1.33; −MTG. imum values of 147%. Contrariwise, gelatine films were offering conspicuous higher tensile strengths to the point of 13.52 MPa in contrast to NaCas-films with maximal values of 4.95 MPa. In general, enzymatic cross-linked NaCas and gelatine films with KNO3 /protein ratios of 1:6 offer the most applicable combination in reference to the total mechanical
performance. The mechanical properties of protein-based films and coatings are chiefly associated with intermolecular and intramolecular interactions inside the protein network depending on arrangement and orientation of the polymer chains (Chambi and Grosso, 2006). The alteration of films mechanical characteristics due to incorporated crystals appears to be related to a weakening of the intermolecular
Fig. 6 – Digital photography exposures of protein films at various potassium nitrate concentrations: (a) 1:4, NaCas; (b) 1:2, NaCas; (c) 1:2, gelatine; (d) 1:1.33, NaCas; −MTG.
1152
chemical engineering research and design 8 8 ( 2 0 1 0 ) 1148–1152
bonds. This results in increasing elongations and decreasing tensile strengths.
3.4.
Morphology
Depending on the potassium nitrate concentration miscellaneous crystal structures and sizes were obtained. As presented in Fig. 5, independent of an enzymatic cross-linking the typical orthorhombic (aragonite-type) structure was observed with KNO3 /NaCas ratios of 1:6 and 1:4 at room temperature. Average crystal sizes between 30 and 200 m were observed at the mentioned concentrations illustrated in Fig. 5(a) and (b). With increasing supersaturation due to the increase of the KNO3 concentration the crystal size increased clearly in conjunction with a structural alternation to a more complicated crystallographic form shown in Fig. 5(c). Brittle columnar sprawling crystals with an effective length up to 100 mm with partial fractures were observed at ratios of 1:1.33. Mentionable differences referring to crystal sizes and structures of NaCas and gelatine films (enzymatically treated and non-treated) could not be ascertained. Protein films with crystallized potassium nitrate at ratios less than 1:6 showed no macroscopic visible crystals in the structure. In these experiments, transparent and smooth structured films were observed. The exposures in Fig. 6(a) show crystalline agglomerates in most instances coexisting with adjacent areas of uniform distributed potassium nitrate crystals in the case of 1:4 ratios. Homogeneously distributed columnar crystals with sizes up to 10 mm were obtained by increasing the KNO3 concentration to ratios of 1:2 (Fig. 6(b)) in NaCas-films. Films made of gelatine show crystalline agglomerates on the surface of the films at ratios of 1:2 (Fig. 6(c)). A further increase of the potassium nitrate concentration (1:1.33, Fig. 6(d)) tends to result in a macroscopic acicular crystalline network after solvent evaporation. The crystals pervaded the entire protein film and protruded at both sides. Under these circumstances the incorporated crystals affect dramatically the overall mechanical performance to the negative. Deviant morphological characteristics of cross-linked films and of samples without enzymatic modification were not ascertained.
4.
Conclusions
This study clearly showed the potential of an inner-film crystallized additive to enhance the mechanical properties of protein-based biodegradable films and coatings as the general basis to expand the application to new fields. Especially, the crystallization of potassium nitrate induced a considerable increase of 122% in the elongation of NaCas-films associated with a minor decrease of film tensile strength. Actually, an improvement of films elongation up to 177% was achieved for crystallized and enzymatic cross-linked gelatine films. However, the great increase of film elongation was accompanied with an explicit decrease of the tensile strength in the case of gelatine. The synergistic effect of potassium nitrate crystallization and enzymatic cross-linking of the protein offered the highest potential in terms of enhancing the overall mechanical performance. Precisely, at KNO3 /NaCas ratios of 1:4 the introduction of cross-linkages increased films tensile strength of 57% and elongation of 137%, respectively. Besides the efforts to improve the physical properties of biodegradable films and coatings additional benefits are able
to facilitate a market share increase or the development of new markets, respectively. Therefore, the selection of reasonable crystallizing additives, e.g. potassium nitrate due to its applications as agricultural fertilizer or for food preservation are promising ways to establish biodegradable films in industry and society.
Acknowledgement The authors want to acknowledge the financial support of the Fachagentur Nachwachsende Rohstoffe e.V. (FNR) for the work presented here.
References Arvanitoyannis, I.S., 2006, Biodegradable edible films and coatings based on protein resources: physical properties and applications in food quality management, In Mallapragada, S.K. and Narasim, B., Narasim, B. (Eds.), Handbook of Biodegradable Polymeric Materials and their Applications (American Scientific Publishers, Stevenson Ranch, USA), pp. 200–235. (American Scientific Publishers, Stevenson Ranch, USA). Audic, J.-L. and Chaufer, B., 2005, Influence of plasticizers and crosslinking on the properties of biodegradable films made from sodium caseinate. European Polymer Journal, 41(8): 1934–1942. Chambi, H. and Grosso, C., 2006, Edible films produced with gelatin and casein cross-linked with transglutaminase. Food Research International, 39(4): 458–466. Clarinval, A.-M. and Halleux, J., 2005, Classification of biodegradable polymers, in Biodegradable Polymers for Industrial Applications, Smith, R. (ed) (Woodhead Publishing Limited, Cambridge, England), pp. 3–29. (Woodhead Publishing Limited, Cambridge, England). Espi, E., Salmeron, A., Fontecha, A., Garcia, Y. and Real, A.I., 2006, Plastic films for agricultural applications. Journal of Plastic Film & Sheeting, 22(2): 85–102. Flanagan, J., Gunning, Y. and FitzGerald, R.J., 2003, Effect of cross-linking with transglutaminase on the heat stability and some functional characteristics of sodium caseinate. Food Research International, 36(3): 267–274. Joo, S.B., Kim, M.N., Im, S.S. and Yoon, J.S., 2005, Biodegradation of plastics in compost prepared at different composting conditions. Macromolecular Symposia, 224: 355–365. Lorenzen, P.C., 2000, Techno-functional properties of transglutaminase-treated milk proteins. Milchwissenschaft-Milk Science International, 55(12): 667–670. Oh, J.H., Wang, B., Field, P.D. and Aglan, H.A., 2004, Characteristics of edible films made from dairy proteins and zein hydrolysate cross-linked with transglutaminase. International Journal of Food Science and Technology, 39(3): 287–294. Siew, D.C.W., Heilmann, C., Easteal, A.J. and Cooney, R.P., 1999, Solution and film properties of sodium caseinate/glycerol and sodium caseinate/polyethylene glycol edible coating systems. Journal of Agricultural and Food Chemistry, 47(8): 3432–3440. Tang, C., Yang, X.Q., Chen, Z., Wu, H. and Peng, Z.Y., 2005, Physicochemical and structural characteristics of sodium caseinate biopolymers induced by microbial transglutaminase. Journal of Food Biochemistry, 29(4): 402–421. Wang, J.S., Zhao, M.M., Yang, X.Q., Jiang, Y.M. and Chun, C., 2007, Gelation behavior of wheat gluten by heat treatment followed by transglutaminase cross-linking reaction. Food Hydrocolloids, 21(2): 174–179. Yi, J.B., Kim, Y.T., Bae, H.J., Whiteside, W.S. and Park, H.J., 2006, Influence of transglutaminase-induced cross-linking on properties of fish gelatin films. Journal of Food Science, 71(9): E376–E383.