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Thermal properties and enzymatic degradation of blends of poly(ε-caprolactone) with starches
Polymer Testing 24 (2005) 756–761 www.elsevier.com/locate/polytest
Material Properties
Thermal properties and enzymatic degradation of blends of poly(3-caprolactone) with starches D.S. Rosa*, D.R. Lopes, M.R. Calil Programa de Po´s-Graduac¸a˜o Stricto Sensu em Engenharia e Cieˆncia dos Materiais, Laborato´rio de Polı´meros Biodegrada´veis e Soluc¸o˜es Ambientais, Universidade Sa˜o Francisco, Rua Alexandre Rodrigues Barbosa, no 45, Centro, CEP 13251-900 Itatiba, SP, Brazil Received 22 February 2005; accepted 30 March 2005
Abstract Waste disposal is an increasing problem as the availability of landfill areas diminishes. Growing environmental concerns have led to the development of alternative biodegradable materials with properties comparable to conventional polymeric materials such as polyethylene and polypropylene. Poly(3-caprolactone) (PCL) is a biodegradable, aliphatic polyester that is attractive because of its availability, variable biodegradation, and good mechanical properties. However, the use of PCL is limited by its high cost. This problem can be overcome by blending PCL with starch, an inexpensive, biodegradable polysaccharide. In this work, we studied the properties of PCL with the cornstarches Amidex 3001 (A1), Amidex 4001(A2) and Penetrose 80 (A3), using PCL/starch proportions of 25/75, 50/50 and 75/25. Thermal analysis of the blends was done by differential scanning calorimetry (DSC) and the susceptibility to enzymatic degradation was assessed in samples of films incubated with 1.0 mg/ml of proteinase K in 0.05 M Tris buffer, pH 8.6, at 37 8C for 72 h. The addition of starch reduced the crystallinity of PCL proportionally to the amount of starch added to the blend (from 0.9 to 39.6%, except for the A350/PCL50 blend for which there was an 8.7% increase in crystallinity). This reduction in crystallinity favored enzymatic degradation by proteinase K, which increased with increasing starch content. However, there was no difference among the starches studied. Light microscopy confirmed that enzymatic digestion affected mainly the starch. q 2005 Elsevier Ltd. All rights reserved. Keywords: Biodegradable polymer; Enzymatic degradation; Poly(3-caprolactone); Starch
1. Introduction In recent years, there has been increasing concern about land filling with non-degradable materials such as plastics. This concern has led to the development of alternative biodegradable materials. Poly(3-caprolactone) (PCL) is a highly flexible, biodegradable, aliphatic polyester [1,2] that is attractive because of its availability, variable biodegradation, and good mechanical properties [3]. However, the use of PCL is limited by its low melting point (w67 8C), which makes it difficult to process PCL by conventional techniques
* Corresponding author. Tel.: C55 11 4534 8046; fax: C55 11 4524 1933. E-mail address: [email protected] (D.S. Rosa).
0142-9418/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymertesting.2005.03.014
used for thermoplastic materials, and by its high cost. This problem can be overcome by blending PCL with starch [3]. To reduce the cost, attempts have been made to blend PCL with various types of starches, an inexpensive, biodegradable polysaccharide [3–6]. Starch is a natural polymer found in granular form in a variety of plants such as potato, corn and cassava [7]. Starch is composed of two glucan chains (amylose and amylopectin). These polymers have the same basic monomer but differ in their length and degree of branching, which ultimately affects their physicochemical properties [8]. Amylopectin consists of granules that are more crystalline, denser and more resistant to penetration by water and to enzymatic action than amylose [9,10]. The chemical structure of starch varies, depending on its origin, and may contain branched chains of various lengths, phosphate
D.S. Rosa et al. / Polymer Testing 24 (2005) 756–761
derivatives and peculiar functional states. These structural and functional variations make starches suitable for many applications [11]. Starch is hydrolyzed to glucose, maltose and maltooligosaccharides by a- and b-amylases and related enzymes [12]. The enzymatic hydrolysis of starch granules may be centripetal (from the surface towards the center of the starch granule) or centrifugal (from the center to the periphery) [13]. Proteinase K normally hydrolyses amide bonds and also attacks ester bonds [14] and is routinely used for experiments in molecular biology. The enzymatic degradation of PCL polymers by proteinase K has been investigated [14–18]. Highly crystalline PCL is totally degraded in 4 days, with the crystallinity decreasing during degradation [15,16]. In this work, we examined the thermal properties and the enzymatic degradation of PCL and blends of PCL with three types of starch.
2. Experimental 2.1. Materials Poly(3-caprolactone) (PCL). PCL was supplied in pellet form by Union Chemical Carbide Ltd (P-767) (Cubata˜o, SP, Brazil) and had a melting index of 1.9G0.3 (ASTM-D1238), a density of 1.145 g/cm3 and a weight–average molecular weight (Mw) of 50,000 g/mol. Corn starch (Amidex 3001) (A1). Regular corn starch was supplied in powder form by Corn Products Brazil Ingredientes Industriais Ltda. (Jundiaı´, SP, Brazil) and contained 27 wt% amylose and 73 wt% amylopectin, with a Mw of 340,000 g/mol and a gelatinization temperature of 90–95 8C. Corn starch (Amidex 4001) (A2). Regular starch of from waxy corn was supplied in powder form by Corn Products Brazil and contained 0–3 wt% amylose and 97–100 wt% amylopectin, with a Mw of 460,000 g/mol and a gelatinization temperature of 90–95 8C. Penetrose 80 (A3). Modified corn starch (obtained by acid hydrolysis), was supplied by Corn Products Brazil and contained 27 wt% amylose and 73 wt% amylopectin, with a Mw of 178,000 g/mol and a gelatinization temperature of 90–95 8C. Proteinase K (R20 U/mg, from Rhizopus sp.; Invitrogen). It was purchased from Sigma Chemical Co. (St Louis, Mo, USA). 2.2. Film preparation The films were prepared by dissolving the materials in 22% (w/v) acetone. Blends of PCL with each type of starch were prepared using PCL/starch proportions (w/w) of 25/75, 50/50 and 75/25. Pure material corresponded to 100/0 for PCL. The solutions were stirred thoroughly at 60 8C and then poured into culture dishes, after which the solvent
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Table 1 Blends of PCL with the starches A1, A2 and A3 Blends denomination
Starch (g)
PCL (g)
A10/PCL100 A125/PCL75 A150/PCL50 A175/PCL25 A20/PCL100 A225/PCL75 A250/PCL50 A275/PCL25 A30/PCL100 A325/PCL75 A350/PCL50 A375/PCL25
0 2.5 5.0 7.5 0 2.5 5.0 7.5 0 2.5 5.0 7.5
10.0 7.5 5.0 2.5 10.0 7.5 5.0 2.5 10.0 7.5 5.0 2.5
was allowed to evaporate in a saturated atmosphere. Table 1 shows how the formulations were prepared. 2.3. Thermal analysis Thermal analyses of PCL were done using a model 204 TASC 414/3A differential scanning calorimeter (DSC) (Netzsch–Gera¨tebau GmbH, Bavaria, Germany) under a nitrogen atmosphere, at a heating rate of 10 8C minK1. Two heating cycles were used for PCL and the blends with starches. The temperatures used were from room temperature to 80 8C for the first heating, and from room temperature to 100 8C for the second heating. The second scan was done using the same heating rate as the first. All DSC experiments were done in duplicate and the thermograms shown refer to the second heating. The crystallinity values were calculated using a heat of fusion (DH0PCL) of 136.1 J/kg for 100% crystalline PCL [19]. 2.4. Enzymatic degradation Each sample was placed in a vial filled with 5 ml of 0.05 M Tris–HCl, pH 8.6, containing 1.0 mg of proteinase K and 0.02% sodium azide. The vials were placed in a thermostatted oven at 37 8C and the buffer/enzyme system was changed every 48 h to maintain the original level of enzymatic activity. After 144 h, the samples were removed from the incubation medium, washed with distilled water, wiped dry, and then weighed and examined by light microscopy. Controls containing PCL and starch alone were prepared in buffer without enzyme. 2.5. Light microscopy The morphology and the degree of phase separation of the PCL/starch blends were assessed by a light microscopy (model XP-500 microscope LABORANA, Sa˜o Paulo, SP, Brazil).
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D.S. Rosa et al. / Polymer Testing 24 (2005) 756–761
Table 2 Melting temperature, temperature of crystallinity, melting heat and crystallinity of pure PCL and the blends with starches A1, A2 and A3 Blends
Melting temperature (8C)
Temperature of crystallinity (8C)
Melting heat (J gK1)
Crystallinity (%)
Pure PCL A125/PCL75 A150/PCL50 A175/PCL25 A225/PCL75 A250/PCL50 A275/PCL25 A325/PCL75 A350/PCL50 A375/PCL25
57.2 59.7 57.2 57.2 59.7 57.2 57.1 59.7 59.6 58.4
20.4 22.9 25.5 25.5 22.9 25.4 22.9 22.9 25.4 22.9
62.8 67.0 49.8 47.4 56.6 52.8 38.0 62.4 68.3 39.1
46.2 49.2 36.6 34.8 41.6 38.8 27.9 45.8 50.2 28.7
3. Results 3.1. Thermal analysis (DSC) Table 2 shows the results of the thermal analysis of PCL and its blends with the starches A1, A2 and A3. Under the DSC conditions used, it was not possible to identify the melting temperatures of the starches because of the low temperature at which decomposition of the starches began [3]. Compared to PCL, the crystallinity of the A125/PCL75 blend increased by 6.5% whereas that of the A150/PCL50 and A175/PCL25 blends decreased by w21 and 15%, respectively. A similar behavior was observed for blends with starches A2 and A3. These results indicated that the greater the amount of starch added to the blend, the greater the decrease in the crystallinity of PCL, from 0.9 to 39.6%, except for the A350/PCL50 blend for which there was 8.7% increase in the crystallinity. This decrease in crystallinity is favorable for enzymatic degradation. The temperature of crystallinity of PCL in the blends increased when compared to pure PCL. In all of the blends containing 25 and 50% starch there was a 12.3 and 24.7% increase in the temperature of crystallization, when compared with that of pure PCL. For blends containing 75% starch (A1, A2, and A3), the increase was 25% for A125/PCL75 and 12.3% for A150/PCL50 and A175/PCL25. The melting temperature of PCL remained the same in all formulations, except for blends containing 25% starch, in which there was an increase of 2.5 8C. The decline in the fusion temperatures
Fig. 1. Photomicrographs of pure PCL. (a) 40!, (b) 100!.
and crystallinity directly reflected the miscibility of the blends [1] and indicated that the mixtures were immiscible [20]. The small decrease in the temperature of fusion caused by starch probably reflected the separation of the two polymeric systems [21]. 3.2. Light microscopy Fig. 1 shows a photomicrograph of PCL in which the PCL spherolits are clearly visible [14]. Fig. 2 shows the photomicrograph of starches A1, A2 and A3 under polarized light. The grains were totally
Fig. 2. Photomicrographs of starches. (a) A1, (b) A1, (c) A2, (d) A2, (e) A3 and (f) A3. (a), (c) and (e) 40!; (b), (d) and (f) K100!.
D.S. Rosa et al. / Polymer Testing 24 (2005) 756–761
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Table 3 Mass retention (%) of pure PCL and the PCL/starches blends treated with proteinase K Blends
Mass retention (%) Control
A10/PCL100 A125/PCL75 A150/PCL50 A175/PCL25 A225/PCL75 A250/PCL50 A275/PCL25 A325/PCL75 A350/PCL50
Proteinase K
Initial
144 h
Initial
48 h
96 h
144 h
100 100 100 100 100 100 100 100 100
100 100 99.4 99.9 100 100 99.6 99.5 100
100 100 100 100 100 100 100 100 100
100 99.8 99.0 20 99.1 98.2 24.5 99.0 97.3
99.4 97.0 95.0 – 96.0 87.3 – 93.5 87.7
98.5 95.0 92.0 – 90.1 66.2 – 85.5 76.9
3.3. Enzymatic degradation
Fig. 3. Photomicrographs of PCL/starch blends. (a) A150/PCL50 without iodine, (b) A150/PCL50 with iodine, (c) A250/PCL50 without iodine, (d) A250/PCL50 with iodine, (e) A350/PCL50 without iodine and (f) A350/PCL50 with iodine. 100! in all cases.
encapsulated, indicating that they were not in gelatinized form. There was no significant variation in the size and form of the grains of the three starches, despite the differences in their content of amylose and amylopectin. For the morphological analysis, blends of 50% starch/50% PCL were chosen because, theoretically, with this ratio, there ought to be no difference in the texture of the phases. However, as shown in Fig. 3, the blends had distinct starch and PCL phases, with a good dispersion of starch in the PCL matrix. The photomicrographs in Fig. 3(a), (c) and (e) show the morphology of blends without the addition of iodine, whereas panels (b), (d) and (f) show the morphology in the presence of iodine. These photomicrographs clearly showed the presence of two distinct phases (starches and PCL). In photomicrographs 3(b) and 3(f) of A1 and A3 with iodine, the shape of a Maltese Cross was clearly visible in the starch grains. However, this phenomenon was not seen with the A2 blend, probably because this starch lacked amylose, which has a linear chemical structure that favors the Maltese Cross formation.
Table 3 shows the percentage retention of mass by pure PCL and its blends with starches after digestion with proteinase K. PCL showed only a 1.5% loss of mass after 144 h incubation with proteinase K. This loss was not significantly different from that seen in the control samples, indicating that PCL was not degraded by this enzyme during this period. Similar results have also been reported by Liu et al. [14] and Mochizuki [18]. The addition of up to 50% A1 starch did not significantly influence the degradability of the blends, with only 5–8% degradation occurring after 144 h in the presence of the enzyme. With similar proportions (up to 50%) of the starches A2 and A3, the extent of degradation was more significant (up to 33%). The greatest degradation occurred in blends containing 75% (w/w) starch (A1, A2, A3); after 48 h, the degradation reached 75–90% (corresponding to a retention of mass of 10–25%). These formulations had low levels of crystallinity (Table 2), which suggested that the degree of degradation depended on this property. Fig. 4 shows pure PCL before and after 144 h of treatment with proteinase K. Although PCL showed a high retention of mass (Table 3), light microscopy revealed significant alterations in the surface of this polymer, including a loss of brightness, the presence of small erosions
Fig. 4. Photomicrographs of the surface of PCL in the presence of proteinase K: before (a) and after (b) a 144 h incubation with proteinase K. 30! in both cases.
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D.S. Rosa et al. / Polymer Testing 24 (2005) 756–761
Fig. 5. Photomicrographs of the surface of blends: A125/PCL75 (a–c) and A150/PCL50 (d–f). (a) and (d) before, (b) and (e) after 96 h, (c) and (f) after 144 h. 30! in all cases.
on the surface, and an increase in the empty spaces between the grains. These results were similar to those reported elsewhere [14,22]. As with pure PCL, the A125/PCL75 and A150/PCL50 blends showed a loss of mass (Table 3), but light microscopy revealed significant alterations in the polymer surface, including a loss of brightness and an altered morphology when compared with samples that were not treated with enzyme for 96 and 144 h (Fig. 5). The greatest changes were seen after 144 h, indicating the onset of degradation by proteinase K. The extent of blend degradation increased with increasing starch content. For the blends with the highest starch concentration, it was not possible to continue enzymatic aging after 48 h, since the samples had already degraded by w80% (Fig. 6); the sample of blend A375/ PCL25 was totally degraded after 48 h of digestion by proteinase K.
Fig. 6. Photomicrographs of samples of control (a) and enzyme treatment (b) samples of A375/PCL25. The samples were incubated without or with proteinase K for 48 h.
Fig. 7 shows photomicrographs of the surface of blends A175/PCL25, A275/PCL25 and A375/PCL25 before and after 48 h of aging with proteinase K enzyme. After 48 h, the surfaces of the samples had a granulated and ‘snowy’ aspect,
Fig. 7. Photomicrographs of the surface of blends A175/PCL25, (a) initial, (b) after 48 h, A275/PCL25 and A375/PCL25 treated with proteinase K. (a), (c) and (e) before incubation; (b), (d) and (f) after 48 h. 30! in all cases.
D.S. Rosa et al. / Polymer Testing 24 (2005) 756–761
indicating that at this starch concentration the biodegradation was intense, probably because of the strong immiscibility of the polymers, as mentioned above. Similar findings have been reported by others [23,24].
4. Conclusions Blends of PCL with three types of starch were viable, despite the incompatibility of the materials that tended to reduce the final crystallinity value of the blend. Light microscopy showed that the blends obtained had a good dispersion of starch in the PCL matrix but formed a twophase system that favored biodegradation. Thermal analysis showed that the presence of starch reduced the crystallinity of PCL. The two-phase system, the degree of crystallinity and the starch content favored enzymatic degradation by proteinase K. However, the type of starch used in the blends had little influence on the properties studied, except for the A250/PCL50 blend, which showed a greater loss of mass after 144 h of aging with proteinase K.
Acknowledgements The authors thank Union Carbide Ltd for supplying PCL and Corn Products Brazil for supplying starches. This work was supported by the Universidade Sa˜o Francisco, FAPESP (Process nos. 99/10716-4 and 02/13202-6) and CNPq (Proc. no. 477942/2003). Derval dos Santos Rosa was supported by CNPq (Proc. no. 303500/2002-6).
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[8] C.J. Slattery, I.H. Kavakli, T.W. Okita, Engineering starch for increased quantity and quality, Trends Plant Sci. 5 (7) (2000) 291. [9] C. Bastioli, Starch–polymer composites in: D. Gilead, G. Scott (Eds.), Degradable Polymers—Principles and Applications, Chapman & Hall, London, 1995, pp. 112–137. (Chapter 6). [10] L.H. Ace, K.N. Hie, K. Nishinari, DSC and rheological studies of the effects of sucrose on the gelatinization and retrogradation of corn starch, Thermochim. Acta 322 (1998) 39–46. [11] J. Jane, Starch properties modifications and applications, in: Ann-Christine Albertsson, Samuel J. Huang (Eds.), Degradable Polymers, Recycling and Plastics Waste Management, Marcel Dekker, New York, 1995, pp. 158–165. [12] E. Sarikaya, T. Higasa, M. Adachi, B. Mikami, Comparation of degradation abilities of a- and b-amylases on raw starch granules, Proc. Biochem. 35 (2000) 711–715. [13] D. French, Organization of starch granules in: R.L. Whistler, J.N. Bemiller, E.F. Parchall (Eds.), Starch, Chemistry and Technology, Academic Press, New York, 1984, pp. 183–247. [14] L. Liu, L. Summing, H. Garreau, M. Vert, Selective enzymatic degradations op Poly(L-lactide) and poly(3-caprolactone), Biomacromolecules 1 (2000) 350–359. [15] Z. Gan, Q. Liang, J. Zhang, X. Jing, Enzymatic degradation of poly(3-caprolactone) film in phosphate buffer solution containing lipases, Polym. Degrad. Stab 56 (1997) 209–213. [16] Z. Gan, D. Yu, Z. Zhong, Q. Liang, X. Jing, Enzymatic degradation poly(3-caprolactone)/poly(DL-lactide) blends in phosphate buffer solution, Polymer 40 (1999) 2859–2862. [17] Y. Oda, A. Yonetsu, T. Urakami, K. Tonomura, Degradation of polylactide by commercial proteases, J. Polym. Environ. 8 (1) (2000) 29–32. [18] M. Mochizuki, M. Hirano, Y. Kanmuri, K. Kudo, Y. Tokiwa, Hydrolysis of polycaprolactone fibers by lipase: effects of draw ratio on enzymatic degradation, J. Appl. Polym. Sci. 55 (1995) 289–296. [19] H. Hatakeyama, T. Yoshida, T. Hatakeyama, The effect of side chain association on thermal and viscoelastic properties. Cellulose acetate based polycaprolactones, J. Therm. Anal. Calorimetry 59 (1) (2000) 157–168. [20] Y.S. Chun, W.N. Kim, Thermal and rheological properties of poly(3-caprolactone) and polystyrene blends, Polymer 41 (2000) 8729–8733. [21] U.S. Ishiaku, K.W. Pang, W.S. Lee, Z.A. Mohd. Ishak, Mechanical properties and enzymatic degradation of thermoplastic and granular sago starch filled poly(3-caprolactone), Eur. Polym. J. 38 (2002) 393–401. [22] N. Gurav, S. Downes, A qualitative in vitro evaluation of degradable materials poly(caprolactone), poly(hydroxybutyrate), poly(hydroxybutyrate)–(hydroxyvalerate) copolymer, J Mater. Sci.: Mater. Med. 5 (1994) 784–787. [23] D.S. Rosa, T.C. Rodrigues, C.G.F. Guedes, M.R. Calil, The effect of thermal ageing on the biodegradation of PCL, PHB-V and their blends with starch in soil compostage, J. Appl. Polym. Sci. 89 (13) (2003) 3539–3546. [24] C.E. Corradini, L.H.C. Mattoso, C.G.F. Guedes, D. Rosa, Mechanical, thermal and morphological properties of poly(3caprolactone)/zein Blends, Polym. Adv. Technol. 93 (2004) 1230–1235.
Material Properties
Thermal properties and enzymatic degradation of blends of poly(3-caprolactone) with starches D.S. Rosa*, D.R. Lopes, M.R. Calil Programa de Po´s-Graduac¸a˜o Stricto Sensu em Engenharia e Cieˆncia dos Materiais, Laborato´rio de Polı´meros Biodegrada´veis e Soluc¸o˜es Ambientais, Universidade Sa˜o Francisco, Rua Alexandre Rodrigues Barbosa, no 45, Centro, CEP 13251-900 Itatiba, SP, Brazil Received 22 February 2005; accepted 30 March 2005
Abstract Waste disposal is an increasing problem as the availability of landfill areas diminishes. Growing environmental concerns have led to the development of alternative biodegradable materials with properties comparable to conventional polymeric materials such as polyethylene and polypropylene. Poly(3-caprolactone) (PCL) is a biodegradable, aliphatic polyester that is attractive because of its availability, variable biodegradation, and good mechanical properties. However, the use of PCL is limited by its high cost. This problem can be overcome by blending PCL with starch, an inexpensive, biodegradable polysaccharide. In this work, we studied the properties of PCL with the cornstarches Amidex 3001 (A1), Amidex 4001(A2) and Penetrose 80 (A3), using PCL/starch proportions of 25/75, 50/50 and 75/25. Thermal analysis of the blends was done by differential scanning calorimetry (DSC) and the susceptibility to enzymatic degradation was assessed in samples of films incubated with 1.0 mg/ml of proteinase K in 0.05 M Tris buffer, pH 8.6, at 37 8C for 72 h. The addition of starch reduced the crystallinity of PCL proportionally to the amount of starch added to the blend (from 0.9 to 39.6%, except for the A350/PCL50 blend for which there was an 8.7% increase in crystallinity). This reduction in crystallinity favored enzymatic degradation by proteinase K, which increased with increasing starch content. However, there was no difference among the starches studied. Light microscopy confirmed that enzymatic digestion affected mainly the starch. q 2005 Elsevier Ltd. All rights reserved. Keywords: Biodegradable polymer; Enzymatic degradation; Poly(3-caprolactone); Starch
1. Introduction In recent years, there has been increasing concern about land filling with non-degradable materials such as plastics. This concern has led to the development of alternative biodegradable materials. Poly(3-caprolactone) (PCL) is a highly flexible, biodegradable, aliphatic polyester [1,2] that is attractive because of its availability, variable biodegradation, and good mechanical properties [3]. However, the use of PCL is limited by its low melting point (w67 8C), which makes it difficult to process PCL by conventional techniques
* Corresponding author. Tel.: C55 11 4534 8046; fax: C55 11 4524 1933. E-mail address: [email protected] (D.S. Rosa).
0142-9418/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymertesting.2005.03.014
used for thermoplastic materials, and by its high cost. This problem can be overcome by blending PCL with starch [3]. To reduce the cost, attempts have been made to blend PCL with various types of starches, an inexpensive, biodegradable polysaccharide [3–6]. Starch is a natural polymer found in granular form in a variety of plants such as potato, corn and cassava [7]. Starch is composed of two glucan chains (amylose and amylopectin). These polymers have the same basic monomer but differ in their length and degree of branching, which ultimately affects their physicochemical properties [8]. Amylopectin consists of granules that are more crystalline, denser and more resistant to penetration by water and to enzymatic action than amylose [9,10]. The chemical structure of starch varies, depending on its origin, and may contain branched chains of various lengths, phosphate
D.S. Rosa et al. / Polymer Testing 24 (2005) 756–761
derivatives and peculiar functional states. These structural and functional variations make starches suitable for many applications [11]. Starch is hydrolyzed to glucose, maltose and maltooligosaccharides by a- and b-amylases and related enzymes [12]. The enzymatic hydrolysis of starch granules may be centripetal (from the surface towards the center of the starch granule) or centrifugal (from the center to the periphery) [13]. Proteinase K normally hydrolyses amide bonds and also attacks ester bonds [14] and is routinely used for experiments in molecular biology. The enzymatic degradation of PCL polymers by proteinase K has been investigated [14–18]. Highly crystalline PCL is totally degraded in 4 days, with the crystallinity decreasing during degradation [15,16]. In this work, we examined the thermal properties and the enzymatic degradation of PCL and blends of PCL with three types of starch.
2. Experimental 2.1. Materials Poly(3-caprolactone) (PCL). PCL was supplied in pellet form by Union Chemical Carbide Ltd (P-767) (Cubata˜o, SP, Brazil) and had a melting index of 1.9G0.3 (ASTM-D1238), a density of 1.145 g/cm3 and a weight–average molecular weight (Mw) of 50,000 g/mol. Corn starch (Amidex 3001) (A1). Regular corn starch was supplied in powder form by Corn Products Brazil Ingredientes Industriais Ltda. (Jundiaı´, SP, Brazil) and contained 27 wt% amylose and 73 wt% amylopectin, with a Mw of 340,000 g/mol and a gelatinization temperature of 90–95 8C. Corn starch (Amidex 4001) (A2). Regular starch of from waxy corn was supplied in powder form by Corn Products Brazil and contained 0–3 wt% amylose and 97–100 wt% amylopectin, with a Mw of 460,000 g/mol and a gelatinization temperature of 90–95 8C. Penetrose 80 (A3). Modified corn starch (obtained by acid hydrolysis), was supplied by Corn Products Brazil and contained 27 wt% amylose and 73 wt% amylopectin, with a Mw of 178,000 g/mol and a gelatinization temperature of 90–95 8C. Proteinase K (R20 U/mg, from Rhizopus sp.; Invitrogen). It was purchased from Sigma Chemical Co. (St Louis, Mo, USA). 2.2. Film preparation The films were prepared by dissolving the materials in 22% (w/v) acetone. Blends of PCL with each type of starch were prepared using PCL/starch proportions (w/w) of 25/75, 50/50 and 75/25. Pure material corresponded to 100/0 for PCL. The solutions were stirred thoroughly at 60 8C and then poured into culture dishes, after which the solvent
757
Table 1 Blends of PCL with the starches A1, A2 and A3 Blends denomination
Starch (g)
PCL (g)
A10/PCL100 A125/PCL75 A150/PCL50 A175/PCL25 A20/PCL100 A225/PCL75 A250/PCL50 A275/PCL25 A30/PCL100 A325/PCL75 A350/PCL50 A375/PCL25
0 2.5 5.0 7.5 0 2.5 5.0 7.5 0 2.5 5.0 7.5
10.0 7.5 5.0 2.5 10.0 7.5 5.0 2.5 10.0 7.5 5.0 2.5
was allowed to evaporate in a saturated atmosphere. Table 1 shows how the formulations were prepared. 2.3. Thermal analysis Thermal analyses of PCL were done using a model 204 TASC 414/3A differential scanning calorimeter (DSC) (Netzsch–Gera¨tebau GmbH, Bavaria, Germany) under a nitrogen atmosphere, at a heating rate of 10 8C minK1. Two heating cycles were used for PCL and the blends with starches. The temperatures used were from room temperature to 80 8C for the first heating, and from room temperature to 100 8C for the second heating. The second scan was done using the same heating rate as the first. All DSC experiments were done in duplicate and the thermograms shown refer to the second heating. The crystallinity values were calculated using a heat of fusion (DH0PCL) of 136.1 J/kg for 100% crystalline PCL [19]. 2.4. Enzymatic degradation Each sample was placed in a vial filled with 5 ml of 0.05 M Tris–HCl, pH 8.6, containing 1.0 mg of proteinase K and 0.02% sodium azide. The vials were placed in a thermostatted oven at 37 8C and the buffer/enzyme system was changed every 48 h to maintain the original level of enzymatic activity. After 144 h, the samples were removed from the incubation medium, washed with distilled water, wiped dry, and then weighed and examined by light microscopy. Controls containing PCL and starch alone were prepared in buffer without enzyme. 2.5. Light microscopy The morphology and the degree of phase separation of the PCL/starch blends were assessed by a light microscopy (model XP-500 microscope LABORANA, Sa˜o Paulo, SP, Brazil).
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Table 2 Melting temperature, temperature of crystallinity, melting heat and crystallinity of pure PCL and the blends with starches A1, A2 and A3 Blends
Melting temperature (8C)
Temperature of crystallinity (8C)
Melting heat (J gK1)
Crystallinity (%)
Pure PCL A125/PCL75 A150/PCL50 A175/PCL25 A225/PCL75 A250/PCL50 A275/PCL25 A325/PCL75 A350/PCL50 A375/PCL25
57.2 59.7 57.2 57.2 59.7 57.2 57.1 59.7 59.6 58.4
20.4 22.9 25.5 25.5 22.9 25.4 22.9 22.9 25.4 22.9
62.8 67.0 49.8 47.4 56.6 52.8 38.0 62.4 68.3 39.1
46.2 49.2 36.6 34.8 41.6 38.8 27.9 45.8 50.2 28.7
3. Results 3.1. Thermal analysis (DSC) Table 2 shows the results of the thermal analysis of PCL and its blends with the starches A1, A2 and A3. Under the DSC conditions used, it was not possible to identify the melting temperatures of the starches because of the low temperature at which decomposition of the starches began [3]. Compared to PCL, the crystallinity of the A125/PCL75 blend increased by 6.5% whereas that of the A150/PCL50 and A175/PCL25 blends decreased by w21 and 15%, respectively. A similar behavior was observed for blends with starches A2 and A3. These results indicated that the greater the amount of starch added to the blend, the greater the decrease in the crystallinity of PCL, from 0.9 to 39.6%, except for the A350/PCL50 blend for which there was 8.7% increase in the crystallinity. This decrease in crystallinity is favorable for enzymatic degradation. The temperature of crystallinity of PCL in the blends increased when compared to pure PCL. In all of the blends containing 25 and 50% starch there was a 12.3 and 24.7% increase in the temperature of crystallization, when compared with that of pure PCL. For blends containing 75% starch (A1, A2, and A3), the increase was 25% for A125/PCL75 and 12.3% for A150/PCL50 and A175/PCL25. The melting temperature of PCL remained the same in all formulations, except for blends containing 25% starch, in which there was an increase of 2.5 8C. The decline in the fusion temperatures
Fig. 1. Photomicrographs of pure PCL. (a) 40!, (b) 100!.
and crystallinity directly reflected the miscibility of the blends [1] and indicated that the mixtures were immiscible [20]. The small decrease in the temperature of fusion caused by starch probably reflected the separation of the two polymeric systems [21]. 3.2. Light microscopy Fig. 1 shows a photomicrograph of PCL in which the PCL spherolits are clearly visible [14]. Fig. 2 shows the photomicrograph of starches A1, A2 and A3 under polarized light. The grains were totally
Fig. 2. Photomicrographs of starches. (a) A1, (b) A1, (c) A2, (d) A2, (e) A3 and (f) A3. (a), (c) and (e) 40!; (b), (d) and (f) K100!.
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Table 3 Mass retention (%) of pure PCL and the PCL/starches blends treated with proteinase K Blends
Mass retention (%) Control
A10/PCL100 A125/PCL75 A150/PCL50 A175/PCL25 A225/PCL75 A250/PCL50 A275/PCL25 A325/PCL75 A350/PCL50
Proteinase K
Initial
144 h
Initial
48 h
96 h
144 h
100 100 100 100 100 100 100 100 100
100 100 99.4 99.9 100 100 99.6 99.5 100
100 100 100 100 100 100 100 100 100
100 99.8 99.0 20 99.1 98.2 24.5 99.0 97.3
99.4 97.0 95.0 – 96.0 87.3 – 93.5 87.7
98.5 95.0 92.0 – 90.1 66.2 – 85.5 76.9
3.3. Enzymatic degradation
Fig. 3. Photomicrographs of PCL/starch blends. (a) A150/PCL50 without iodine, (b) A150/PCL50 with iodine, (c) A250/PCL50 without iodine, (d) A250/PCL50 with iodine, (e) A350/PCL50 without iodine and (f) A350/PCL50 with iodine. 100! in all cases.
encapsulated, indicating that they were not in gelatinized form. There was no significant variation in the size and form of the grains of the three starches, despite the differences in their content of amylose and amylopectin. For the morphological analysis, blends of 50% starch/50% PCL were chosen because, theoretically, with this ratio, there ought to be no difference in the texture of the phases. However, as shown in Fig. 3, the blends had distinct starch and PCL phases, with a good dispersion of starch in the PCL matrix. The photomicrographs in Fig. 3(a), (c) and (e) show the morphology of blends without the addition of iodine, whereas panels (b), (d) and (f) show the morphology in the presence of iodine. These photomicrographs clearly showed the presence of two distinct phases (starches and PCL). In photomicrographs 3(b) and 3(f) of A1 and A3 with iodine, the shape of a Maltese Cross was clearly visible in the starch grains. However, this phenomenon was not seen with the A2 blend, probably because this starch lacked amylose, which has a linear chemical structure that favors the Maltese Cross formation.
Table 3 shows the percentage retention of mass by pure PCL and its blends with starches after digestion with proteinase K. PCL showed only a 1.5% loss of mass after 144 h incubation with proteinase K. This loss was not significantly different from that seen in the control samples, indicating that PCL was not degraded by this enzyme during this period. Similar results have also been reported by Liu et al. [14] and Mochizuki [18]. The addition of up to 50% A1 starch did not significantly influence the degradability of the blends, with only 5–8% degradation occurring after 144 h in the presence of the enzyme. With similar proportions (up to 50%) of the starches A2 and A3, the extent of degradation was more significant (up to 33%). The greatest degradation occurred in blends containing 75% (w/w) starch (A1, A2, A3); after 48 h, the degradation reached 75–90% (corresponding to a retention of mass of 10–25%). These formulations had low levels of crystallinity (Table 2), which suggested that the degree of degradation depended on this property. Fig. 4 shows pure PCL before and after 144 h of treatment with proteinase K. Although PCL showed a high retention of mass (Table 3), light microscopy revealed significant alterations in the surface of this polymer, including a loss of brightness, the presence of small erosions
Fig. 4. Photomicrographs of the surface of PCL in the presence of proteinase K: before (a) and after (b) a 144 h incubation with proteinase K. 30! in both cases.
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Fig. 5. Photomicrographs of the surface of blends: A125/PCL75 (a–c) and A150/PCL50 (d–f). (a) and (d) before, (b) and (e) after 96 h, (c) and (f) after 144 h. 30! in all cases.
on the surface, and an increase in the empty spaces between the grains. These results were similar to those reported elsewhere [14,22]. As with pure PCL, the A125/PCL75 and A150/PCL50 blends showed a loss of mass (Table 3), but light microscopy revealed significant alterations in the polymer surface, including a loss of brightness and an altered morphology when compared with samples that were not treated with enzyme for 96 and 144 h (Fig. 5). The greatest changes were seen after 144 h, indicating the onset of degradation by proteinase K. The extent of blend degradation increased with increasing starch content. For the blends with the highest starch concentration, it was not possible to continue enzymatic aging after 48 h, since the samples had already degraded by w80% (Fig. 6); the sample of blend A375/ PCL25 was totally degraded after 48 h of digestion by proteinase K.
Fig. 6. Photomicrographs of samples of control (a) and enzyme treatment (b) samples of A375/PCL25. The samples were incubated without or with proteinase K for 48 h.
Fig. 7 shows photomicrographs of the surface of blends A175/PCL25, A275/PCL25 and A375/PCL25 before and after 48 h of aging with proteinase K enzyme. After 48 h, the surfaces of the samples had a granulated and ‘snowy’ aspect,
Fig. 7. Photomicrographs of the surface of blends A175/PCL25, (a) initial, (b) after 48 h, A275/PCL25 and A375/PCL25 treated with proteinase K. (a), (c) and (e) before incubation; (b), (d) and (f) after 48 h. 30! in all cases.
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indicating that at this starch concentration the biodegradation was intense, probably because of the strong immiscibility of the polymers, as mentioned above. Similar findings have been reported by others [23,24].
4. Conclusions Blends of PCL with three types of starch were viable, despite the incompatibility of the materials that tended to reduce the final crystallinity value of the blend. Light microscopy showed that the blends obtained had a good dispersion of starch in the PCL matrix but formed a twophase system that favored biodegradation. Thermal analysis showed that the presence of starch reduced the crystallinity of PCL. The two-phase system, the degree of crystallinity and the starch content favored enzymatic degradation by proteinase K. However, the type of starch used in the blends had little influence on the properties studied, except for the A250/PCL50 blend, which showed a greater loss of mass after 144 h of aging with proteinase K.
Acknowledgements The authors thank Union Carbide Ltd for supplying PCL and Corn Products Brazil for supplying starches. This work was supported by the Universidade Sa˜o Francisco, FAPESP (Process nos. 99/10716-4 and 02/13202-6) and CNPq (Proc. no. 477942/2003). Derval dos Santos Rosa was supported by CNPq (Proc. no. 303500/2002-6).
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