Bombyx mori Silk Fibroin Composites

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Purified silk fibroin was dissolved in triple aqueous solution (CaCl2/C2H5OH/H2O=1/2/8), and then. SF aqueous solution (the concentration is 3 wt%) was ...

Advanced Materials Research Vols. 306-307 (2011) pp 894-900 Online available since 2011/Aug/16 at www.scientific.net © (2011) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.306-307.894

Natural Rubber/Bombyx mori Silk Fibroin Composites: Preparation and Properties Wang Qinghuang1, a, Luo Yongyue2, b, Yi Zhifeng3,c , Feng Chunfang3,d , Wang Yueqiong1,e and Peng Zheng1,f 1

State Engineering and Technology Research Center for Key Tropical Crops. , Haikou 571101, China; 2

3

Chinese Agricultural Ministry Key Laboratory of Tropical Crop Product Processing, Agricultural Product Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524001, China

Centre for Material and Fibre Innovation, Institute for Technology Research Innovation, Deakin University, Geelong Vic 3217, Australia a d

[email protected],b [email protected], c [email protected],

[email protected], [email protected], f [email protected]

Keywords silk fibroin natural rubber antibacterial property

Abstract A novel natural rubber/silk fibroin (NR/SF) antibacterial composite was prepared firstly by using bombyx mori silk fibroin as the antimicrobial filler through latex compounding techniques. The results from scanning electron microscopy and universal tensile machine proved that the compatibility between SF and NR matrix is excellent, and the tensile strength of composite sheets is enhanced to some extent. NR/SF antibacterial composite sheets have a good antimicrobial effect on Escherichia coli and Staphylococcus aureus. Moreover, the suitable loading of SF is 0.5~1 wt% according to the results above. Introduction Natural rubber (NR) latex possesses many specific features which provide widespread applications in using rubber products. Particularly, in the trade of medical health NR is made into latex gloves, condoms, blood transfusion tubes, Foley catheters and so on. Alternatively, due to the poor antibacterial property of NR products, many kinds of disease-causing bacteria easily breed on the surface of products. For instance, feculent latex gloves, the most commonly used NR products in hospitals, may cause the wound of the patient infected which could in turn lead to associated complaint. One of the conventional Hospital Acquired Infection is caused by indwelling catheters including indwelling urinary catheter and central venous catheter [1]. Therefore, endowing NR material with antibacterial property will intensify the extension of applications. Introducing the antimicrobial agent can bring about a good antibacterial property to NR materials. Recently, inorganic and organic antimicrobial agents have been used as antibacterial fillers in wide area [2-4]. With the development of technology, natural antimicrobial agents, as novel organic agents, are increasingly attracted many attention of researchers because of their All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP, www.ttp.net. (ID: 128.184.132.243-17/10/11,01:50:25)

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natural biocompatibility [5-7]. Silk fibroin (SF), among all kinds of natural antibodies, is a type of protein generated from Bombyx mori (silkworms). It mainly consists of the amino acids glycine, alanine, and serine, which form antiparallel β sheets in the spun fibers [8-10]. Furthermore, SF can restrain the breeding of regular bacterial to a large extent [11]. Therefore, in this paper natural rubber/silk fibroin (NR/SF) antibacterial composite was prepared using bombyx mori silk fibroin as the antimicrobial filler via latex compounding techniques for the first time. Materials and Method Chemicals The ammonia-preserved natural rubber latex (NRL) with a total solid content of 60% was purchased from Qianjin State Farm, Guangdong province; Grade 5A cocoons of Bombyx mori silkworm were provided by Zhanjiang Qinsheng Co., Guangdong province. Escherichia coli (EC) and Staphylococcus aureus (SA) were obtained from National Culture Collection, and culturing codes were ATCC 25922 and ATCC 6538 respectively. The culture medium contained nutrient broth (NB), nutrient agar (NA) and tube slant culture medium. Anhydrous CaCl2, NaCO3, ethanol absolute and other reagents were analytical grade and used as received. Preparation of NR/SF composites Purified silk fibroin was dissolved in triple aqueous solution (CaCl2/C2H5OH/H2O=1/2/8), and then SF aqueous solution (the concentration is 3 wt%) was added into 50g of NR latex. Subsequently, the distilled water was complemented into the mixing emulsion, so that the total solid content of the composite emulsion was 30%. The dry weight of SF/NR ratio was adjusted to 1wt%, 2wt%, 3wt% and 5wt% respectively. To be compared NR latex with total solid content 30% was the blank. All of the composite emulsion was stirred for 12 hours at room temperature. Eventually, composite films were obtained via drying on glass plate. The composite emulsion was noted as NR, NS-0.5, NS-1, NS-2, NS-3 and NS-5, and the composite sheets were noted as NR/SF-0, NR/SF-0.5, NR/SF-1, NR/SF-2, NR/SF-3 and NR/SF-5. Results and Discussions The measurement of UV-Vis absorption As a fibrous protein, SF possesses characteristic absorption peak of the general protein in ultraviolet region (Figure 1). The range of 200 ~ 219 nm absorption segment, below 250 nm mainly is caused by the peptide bond of n→π* electron transition [12], and the π→π* electron transition in the aromatic group of side chain bring about the absorption peak of 275 nm, above 250 nm [13]. It can be predicted that in the structure of SF there are large numbers of aromatic groups in the side chains. After added into NR latex, the ultraviolet absorption intensity of the latex is enhanced by SF (Figure 2). The absorbance of maximum absorption peak of NR latex is 0.32 at 219 nm, while the absorption intensity surpasses 0.55 entirely due to a hyper-chromic effect of SF at the maximum absorption wavelength. In addition, before the absorption intensity decreases at NR/SF-3, it goes up to the peak at NR/SF-2 (absorbance 0.69). The structure of SF in 2 wt% composite emulsion has probably changed, resulting in the reduction of hyper-chromic function. As a consequence, this structure changing may influence the properties of dried composite sheets, which will be discussed in the following sections.

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4.0

200~219 nm 3.5

Absorbance / a.u.

3.0 2.5 2.0 1.5

275 nm 251 nm

1.0 0.5 0.0 200

220

240

260

280

300

320

340

360

380

400

Wavelength / nm

Figure 1 Ultraviolet visible absorption spectra of SF solution. 0.7

NS-0 NS-0.5 NS-1 NS-2 NS-3 NS-5

Absorbance / a.u.

0.6 0.5 0.4 0.3 0.2 0.1

219 nm 200

300

400

500

600

Wave length / nm

Figure 2 Ultraviolet visible absorption spectra of NR latex with different SF loadings.

Antibacterial Property of NR/SF composite sheets Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC6538) were selected as the experimental strain, and the initial concentration of Escherichia coli and Staphylococcus aureus were 5.2×105cfu/mL and 10.0×105cfu/mL respectively. Subsequently all the sheet composites were cut into (50±2)mm×(50±2)mm, inoculated by bacteria solution, covered with the membrane and cultured in Petri dish for 24 h. After the incubation all the samples were rinsed completely with the eluent and the total colony of bacterial was recorded by counting through the microscope. All the results were showed in Table 1. Table 1 Antibacterial properties of NR sheets with different SF loadings Concentration /wt%

Echerichia coli

Staphylococcus

Total colony/ cfu/mL

Inhibition /%

Total colony/ cfu/mL

Inhibition /%

NR/SF-0

4.3×105

0

12.0×105

0

NR/SF-0.5

0.2×105

95.3

2.2×105

81.3

NR/SF-1

0.4×105

90.7

4.5×105

62.5

NR/SF-2

2.4×105

44.2

8.7×105

27.1

NR/SF-3

5.6×105



14.5×105



NR/SF-5

4.6×105



4.7×105

60.4

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Inhibitory rate does not increase accompanying with the quantity of SF. When the content of SF is more than 3 wt% (NR/SF-3 and NR/SF-5), rubber sheets do not possess antibacterial properties no longer, and even the number of living bacterial in NR/SF composites is higher than that in blank NR samples. Furthermore, it can be seen from Table 1 that, when the loading of SF is 0.5 wt%, for these two microbes, Echerichia coli and Staphylococcus, the inhibition rates reach to 95.3% and 81.3% respectively, which is much higher than other samples. Therefore, the suitable ratio of SF/NR should be 0.5 ~ 1 wt%. The morphology of cross-section of NR/SF composite sheet A

B

C

Figure 3 SEM images of the transect of NR sheets with different SF loadings A: NR/SF-0; B: NR/SF-1; C: NR/SF-5

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The composite sheets were immersed in liquid nitrogen and then broken to produce fracture surface specimens. The fracture surface of cross-section can be observed by SEM. NR, NR/SF-1 and NR/SF-5 have been chosen as the model materials in order to study the distribution of SF in NR matrix. The cross-section of pure NR presents homogeneous rubber phase (Figure 3A). Layer-liked SF solid mentioned in reference [14] can be obviously observed when adding a certain amount of SF. Interestingly, when the content is lower (0.5 wt%), SF performs aggregating in a large bulk in NR matrix (Figure 3B). That may be because the extending situation is not stable for SF, the fibrous protein, as a result it tends to be the state of the lowest energy, which will lead to the aggregation. However, when the loading is higher (5 wt%), the distribution of SF throughout NR matrix is uniform (Figure 3C). Moreover SEM image describes evident two-phase, SF phase and NR phase. And the aggregation of SF decreases. Therefore SF has excellent compatibility with NR matrix. Mechanical properties of composite films In this paper all the rubber sheets are not vulcanized by sulfur curing system. It can be seen from Table 2 that the tensile strength of pure NR sheets is 1.31MPa, but there is an obvious improvement to 3.50MPa, when adding 0.5wt% SF. Moreover with the increase of SF loadings the tensile strength gradually rises except for a slight decrease at NR/SF-1. The highest tensile strength is 5.03MPa when the amount of SF is 5wt%, which might result in the excellent distribution of SF in NR matrix (Figure 3C). Overall, SF can improve the mechanical property of NR materials to some extent. Incorporating the antibacterial property (Table 1) the ideal addition should be about 0.5~1wt%. Table 2 Mechanical property of sheet composite with different SF content Samples

NR/SF-0

NR/SF-0.5

NR/SF-1

NR/SF-2

NR/SF-3

NR/SF-5

Tensile strength (MPa) Tensile modulus (MPa) 300% elongation 500% elongation 700% elongation Elongation at break (%)

1.31

3.50

2.87

3.90

4.48

5.03

0.47 0.52 0.84 806

0.51 0.58 1.09 957

0.55 0.65 1.24 894

0.67 0.88 1.82 886

0.88 1.37 3.19 789

1.25 2.48 — 662

The exploration of the antibacterial structure in composites Due to the similar absorption peak of –OH and –NH2, before FTIR measurement, all samples are dried sufficiently to decline the effect of -OH at 3300 cm-1. As can be seen from Figure 4, the intension of the absorption peak caused by -NH2 gradually goes up at the wave number of 3300 cm-1 accompanying with the increasing amount of SF. The absorption peaks at 1660 cm-1 and 1540 cm-1 represent the structure of amide I and amide II in protein. It can be seen from Figure 4 that when the content of SF is 2wt%, the absorption peak appeals to divide into two peaks near 1660 cm-1, which demonstrate the possibility of structure changing related to amide I. Moreover, as mentioned before the antibacterial property of composite sheets deteriorates sharply at this SF content (Table 1). Therefore, it can be predicted that the antibacterial target of SF in NR matrix is more likely to be associated with the structure of amide I in SF.

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SFA0

SFB0.5

SF 1

C

SFD2

T% %T

SFE3 SF 5

F

4000.0

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

400.0

cm-1 cm-1

Figure 4 FTIR spectra of NR sheets with different content of SF. A: NR/SF-0, B: NR/SF-0.5, C: NR/SF-1, D: NR/SF-2, E: NR/SF-3, F: NR/SF-5. Conclusions SF has been successfully used as an antibacterial agent to prepare natural rubber/silk fibroin composites. The results show that the antibacterial property of composite sheets reaches to 95.3% for Echerichia coli and 81.3% for Staphylococcus respectively. The tensile strength of composites with different SF loadings increases to some extent. To sum up from the antimicrobial property and tensile performance, the optimum addition of SF is 0.5wt% according to dry rubber. Furthermore, it can be predicted that the antibacterial group may target to the structure of amide I in SF protein. Acknowledgements National Program on Key Basic Research Project of China (973 Program)(contract grant number: 2010CB635109)and Ministry of Science and Technology R & D research institutes special fund contract grant number: 2008EG134285)are gratefully acknowledged. References [1] E. Mangini, S. Segal-Maurer, J. Burns, A. Avicolli, C. Urban, N. Mariano, L. Grenner, C. Rosenberg and J. J. Rahal: Infect. Control. Hosp. Epidemiol. Vol. 28, (2007), p. 1261. [2] W. Kong, C. Jin, X. Xiao, Y. Zhao, Z. Li, P. Zhang, W. Liu and X.F. Li: J. Hazard. Mater. Vol. 179 (2010), p. 742. [3] J.H. Sim, C.H. Khoo, L.H. Lee and Y.K. Cheah: J. Microbiol. Biotechnol. Vol. 20 (2010), p. 651. [4] S. Bera, G.G. Zhanel and F. Schweizer: J. Antimicrob. Chemother. Vol. 65 (2010), p. 1224.

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[5] T.R. Stratton, J.A. Howarter, B.C. Allison, B.M. Applegate and J.P. Youngblood: Biomacromolecules. Vol. 11 (2010), p. 1286. [6] K. Madhumathi, P.T. Sudheesh Kumar, S. Abhilash, V. Sreeja, H. Tamura, K. Manzoor, S.V. Nair and R. Jayakumar: J. Mater. Sci. Mater. Med. Vol. 21 (2009), p. 807. [7] M.M. Henricus, K.R. Fath, M.Z. Menzenski and I.A. Banerjee: Macromol. Biosci. Vol. 9 (2009), p. 317. [8] A.S. Gobin, V.E. Froude and A.B. Mathur: J. Biomed. Mater. Res. Vol. 74 A (2005), p. 465. [9] S.W. Ha, A.E. Tonelli and S.M. Hudson: Biomacromol. Vol. 6 (2005), p. 1722. [10] W. Tao, M.Z. Li and C.X. Zhao: Int. J. Biol. Macromol. Vol. 40 (2007), p. 472. [11] Y.Q. Zhang, W.D. Shen, R.L. Xiang, L.J. Zhuge, W.J. Gao and W.B. Wang: J. Nanopart. Res. Vol. 9 (2007), p. 885. [12] N. Sreerama and R.W. Woody: Methods Enzymol. Vol. 383 (2004), p. 318. [13] D.M. Rogers and J.D. Hirst: Chirality.Vol. 16 (2004), p. 234. [14] G. Freddi, G. Pessina and M. Tsukada: Int. J. Biol. Macromol. Vol. 24 (1999), p. 251.

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Natural Rubber/Bombyx Mori Silk Fibroin Composites: Preparation and Properties 10.4028/www.scientific.net/AMR.306-307.894