Bombyx Mori Silk Fibers Released from Cocoons by Alkali Treatment

Journal of Life Sciences and Technologies Vol. 3, No. 1, June 2015

Mechanical Properties and Biocompatibility of Attacus atlas and Bombyx mori Silk Fibers Released from Cocoons by Alkali Treatment

Tjokorda Gde Tirta Nindhia and I. Wayan Surata Department of Mechanical Engineering, Udayana University, Jimbaran, Bali, Indonesia, 80361 Email: [email protected]

Zdeněk Knejzlík and Tomáš Ruml Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 5, 166 28, Prague, Czech Republic

Tjokorda Sari Nindhia Faculty of veterinary Madicine, Udayana University, Jl. P.B. Sudirman, Denpasar, Bali, Indonesia, 80114

Abstract—Natural silks, produced by spiders and insects, historically used in textile industry [6]. Fibers present in represent perspective source of biomaterials for the cocoons are mainly composed from fibroin complex regenerative medicine and biotechnology because of their [7] produced from paired labial glands [8]. About 10 – 12 excellent biocompatibility and physico-chemical properties. μm fibroin fibers in B. mori cocoon are tethered by It was previously shown that silks produced by several amorphous protein; sericin, which can be released from members of Saturniidae family have excellent properties in comparison to silk from B. mori, the most studied silkworm. cocoon by washing in mild alkali conditions or hot water, Efficient degumming of silk fibers is a critical step for a process, designated as degumming [9]. Fine fibers, subsequent processing of fibers and/or fibroin. In this study, obtained by degumming, can be used as source of fibroin we describe cheap, environmentally friendly and efficient which may be next solubilized in denaturing agents such NaOH-based degumming of A. atlas fibers originated from as highly concentrated solution of lithium salts, calcium natural cocoon. We found that 100 mM NaOH nitrate or organic ionic liquids. Solubilized fibroin may concentration was efficient for complete A. atlas cocoon be regenerated into desired template structures such as disintegration to individual fibers; while in case of B. mori, fibers, layers or sponges [10]. However, there are studied near complete hydrolysis was observed at the same members of insect producing wild silk. Several studies concentration as shown SDS-PAGE and weight analysis. EM analysis revealed that 100 mM NaOH treatment lead to showed that natural silks have unique properties in complete degumming of A. atlas fibers whilst in case of B. comparing with B. mori silk. It was shown that fibroin mori it was observed at 10 mM NaOH. These observations from Antheraea pernyi, member of Saturniidae family, are in agreement with the tensile analysis showing higher which belongs to an order Lepidoptera has better tensile strength for NaOH degummed fibers over the fibers properties for cultivation of feline fibroblast cells obtained by degumming in hot distilled water. Both types of compared to B. mori [11]. Similarly, matrices prepared degummed silks fibers are usable for cultivation of human from Antheraea mylitta fibroin enable efficient osteosarcoma cells what indicates their prospective attachment and growing of postnatal rat cardiomyocytes biomedical application. [12]. Unique attachment properties of A. mylitta fibroin  Index Terms—attacus atlas; bombyx mori, degumming, matrices could be connected with natural occurrence of cocoon, silk short amino acid RGD motif [13] in its molecule. Main attractiveness of silks produced by members of Saturniidae is linked not only with their excellent I. INTRODUCTION biocompatibility. They also have different mechanical Natural silks, produced by many species of insects and properties in comparison with spider of Nephila species spiders, represent an attractive material for various and B. mori [14]. However, it is known that conditions biotechnological and biomedical applications [1]-[3]. during degumming could influence fiber silk properties They have unique properties such as high strength and [15], [16]. biocompatibility [4], [5]. Most studied insect silk is Here we show that cocoons of A. atlas (Fig. 1) can be obtained from cocoons of silkworm Bombyx mori. This used as a source of fine fibers, which can be prepared by silkmoth species was domesticated more than 4,000 years using NaOH solution for degumming. The resulting ago and fibers isolated from its cocoons have been fibers are biocompatible with mammalian cells as shown by experiment where they served as a support material for Manuscript received February 5, 2015; revised July 14, 2015. cultivation of human osteosarcoma cell line U2OS.

boiled for 1 h. In the case of A. atlas, only middle parts of the cocoons were used. The degumming solution was then removed and residual mechanical particles were pelleted by centrifugation at 20,000 x g for 10 min. Supernatant was dialyzed against deionized water overnight through cellulose membrane (cut-off 1,000). No precipitate was observed in any case. Dialyzed supernatant was mixed with Laemli buffer in a ratio 1:1, and protein content in 25 l was analyzed by using 10 % Tris-Tricine SDS-PAGE with following detection using Coomassie blue. D. Scanning Electron Microscopy (SEM) Weighted differently degummed pieces of A. atlas and B. mori cocoons were attached by scotch tape on a stage cylinder and they were sputter coated with gold. SEM analysis was carried out at 15 kV and image was recorded by using digital camera.

E. Cell Cultivation on the Fibers and Fluorescence Figure 1. The metamorphose of the A. Atlas silkmoth. (A). Eggs, (B) Caterpillar, (C) Cocoon, (D). A. Atlas silkmoth Microscopy A. atlas cocoons were boiled in 0.1 M NaOH for 1 h and released fibers were extensively washed by hot water. II. METHODOLOGY Unwoven 50 mg of fibrous tufts were sterilized with 70 % ethanol for 12 h at room temperature. Samples were A. Cocoon NaOH Treatment and Weight Analysis washed four times by PBS and then an excess of liquid Cocoons of B. mori or A. atlas were obtained from was removed by a vacuum tip to semidry state. Fibrous local cocoon collectors in Yogyakarta, Central Java, tuft was soaked with suspension of U2OS cells in DMEM Indonesia. The medium shell parts of raw cocoons were cultivation medium supplemented with 10 % FBS 5 cut to pieces of average weight approximately 25 – 35 mg. (10 cells per ml) for 2 h in the atmosphere of 5 % CO2 at Each weighted piece was incubated in 10 ml of water or 37°C with 95 % humidity. Next, the excess of the cell NaOH solution in concentration range from 0.001 to 1 M suspension was gently aspirated and the fibers were and boiled for 1 h. In 10 min periods, the samples were entirely immersed in the cultivation medium and the cells vigorously shaken for 20 s. Insoluble materials was then were cultivated for six days at the same conditions collected on pre-weighted 0.45 mm nitrocellulose described above. To visualize the cells, the fibers were membrane by vacuum filtration and intensively washed removed from the cultivation medium, transferred to PBS by 70°C water. Membranes with insoluble material were and gently washed twice with PBS. Cells attached to the dried on air and then incubated in desiccator with silica fibers were fixed with 4 % formaldehyde in PBS for 20 gel for 24 h and then weighted. Percent of insoluble min and washed twice. Nuclei were stained by using 1 material after NaOH treatment was computed by formula g/ml DAPI (Sigma-Aldrich, USA) and actin (mt/mcp) x 100, where mt is the weight of residual microfilaments by 10 g/ml phalloidin covalently material after degumming, filtering and drying and mcp is conjugated with TRITC (Sigma-Aldrich, USA) in PBS the original weight of the cocoon piece. for 20 min at room temperature. Fluorescence microcopy was carried by microscope IX-71 (Olympus, Japan). B. Tensile Analysis

The tensile test was carried out as described and III. RESULT AND DISCUSION illustrated elsewhere [17]-[19]. Single fibers about 5 cm long were obtained from degummed material and were A. Effect of Alkaline Treatment on B. Mori and A. Atlas glued across cardboard frame with distance length 30 cm Cocoons as defining a gauge length. The tensile test was Whole raw B. mori and A. atlas cocoons cuts were performed with screw test stand (ALX-S, INA) at a incubated either in boiling water or in NaOH solutions in constant cross head speed. A balance (Gewinn, INA, concentration ranging from 0.001 to 1 M for1 h. B. mori resolution ± 10 mg) attached to the lower end of the cocoons were visibly disintegrated in 0.01 M NaOH sample was used to measure load as replacement of conventional load cell. The test was performed under while at higher NaOH concentrations, the released fibers conditions of 30oC and 60% relative humidity. were broken into fragments observable by naked eye (Fig. 2A). The A. atlas cocoon integrity was moderately C. Analysis of Released Proteins after Degumming by affected by treatment with NaOH at 0.1 M concentration; SDS-PAGE when compact fibers were released. This process was 100 mg of cocoon raw material was incubated in 1 ml accompanied by a formation of brownish extract (Fig. of NaOH in a concentration range from 0 to 1 M for and 2A). Evaluation of the yield of alkali degumming by

weight analysis showed much higher sensitivity of B. to detect any released protein from A. atlas cocoons mori cocoons to NaOH treatment compared to A. atlas subjected to degumming with NaOH in concentration ones (Fig. 2B). We observed release of about 15% range 0 – 0.01 M (Fig. 2C, line 6 – 8). Removal of about material in water. However, efficient degumming 15% amount of material (Fig. 2B) detectable as faint occurred only when B. mori cocoons were treated with smear on the SDS PAGE gel occurred only after 0.001 or 0.01 M NaOH, which resulted in release of treatment with0.1 and 1M NaOH (Fig. 2C, line 9 and 10); about 25 % of material (by weight). Higher NaOH however, degumming by 0.1 M NaOH was clearly concentrations caused considerable material release up to efficient (Fig. 2A). values corresponding to 75 % and 98 % of original B. Impact of Alkaline Treatment on Fibers Morphology weight at 0.1 and 1 M NaOH, respectively. We observed and Tensile Properties approximately 5 % weight loss of A. atlas cocoons even during treatment in water or NaOH at concentrations up Next, we investigated the effect of 0.01 and 0.1 M to 0.01 M. The weight decrease, of about 18 % material NaOH treatment on morphology of the fibers by scanning during 0.1 M NaOH treatment, well corresponded with electron microscopy (SEM, Fig. 3). For B. mori, efficient the fibers duo to their release from the cocoons (Fig. 3A). removal of the sericin coat was observed after treatment by 0.01 M NaOH, which well corresponded with releasing fine fibers. The degumming with 0.1 M NaOH resulted in strong hydrolysis of fibers (Fig. 2A) and SEM analysis showed residual material of rather disorganized fiber morphology.

Figure 2. Effect of the NaOH treatment on B. mori and A. atlas cocoons. Figure 3. Electron microscopic analysis of degummed cocoon (A) Effect of NaOH treatment on A. atlas cocoon morphology (B) Weight analysis of cocoons after NaOH degumming (n=4). (C) SDS- Raw A. atlas cocoons contained visible extra fibrillar PAGE analysis of proteins released during NaOH degumming material which was almost entirely removed during the We used SDS-PAGE analysis to determine character NaOH treatment at concentrations ranging from 0.01 to of proteins released during degumming process of 0.1 M. Tensile properties of B. mori and A. Atlas fibers cocoons at different NaOH concentration (Fig. 2C). In the obtained from both degumming protocols are presented in case of B. mori, the protein bands were detected already Fig. 4 as stress-strain curves, based on average values after water treatment, which is in correlation to the shown in Table I and Table II. It should be noted that the aforementioned weight loss. As expected, the content of tensile strength (u) values were higher for degumming released proteins increased when 0.001 M NaOH was protocols using alkali treatment. The failure strain (u) for used instead of water (Fig. 2C, line 1 and 2). On the other both types of silk were similar for degumming both in hand, we observed massive hydrolysis at NaOH water and NaOH solution. The elastic modulus (E) for concentrations exceeding 0.001 M. This phenomenon degumming using NaOH treatment was higher compared was clearly visible at 0.1 M NaOH treatment which with the result obtained with boiling water as degumming resulted in degradation of the released proteins to those of medium. molecular weights ranging within an interval of 4 – 14 C. Cell Growth on Degummed Fibers kDa (Fig. 2C, line 4). It is necessary to remark that the lower signal of Coomassie blue stained proteins present Finally, we analyzed biocompatibility of the fibers in extracts obtained by using 0.1 M NaOH was also obtained by degumming of A. atlas and B. mori cocoons affected by lower efficiency of staining the shorter by using 0.1 M NaOH and 0.01 M, respectively (Fig. 5). proteins due to their lower dye binding affinity and less The fibers were sterilized by 70% ethanol and a efficient fixation of such proteins in the gel. In the case of suspension of human osteosarcoma cell line (U2OS) was 1 M NaOH treatment (Fig. 2C, line 5), no signal was applied. The cells were able to attach and grow during detected probably due to a formation of very short following six days. For better visibility were the cell hydrolyzed peptide fragments, which could not be nuclei stained with DAPI. Fig. 5 demonstrates that both B. resolved on SDS-PAGE. In contrast to B. mori, we failed mori and A. atlas fibers released from cocoons by NaOH

treatment were fully covered with U2OS cells, indicating be used for efficient degumming of A. atlas fibers. their excellent biocompatibility. In both cases, the cells Namely, fine individual fibers suitable for cell cultivation were attached and grew well on the fiber surface. could be obtained by 0.1 M NaOH treatment. It was Seemingly higher colonization and cell growth on the A. reported previously, that cocoons of A. atlas and also of atlas fibers may be explained by their larger radius other members of Saturniidae family contain high compared to the B. mori fibers. amount of calcium oxalate, which is removable by 1 M EDTA [e.g. 20]. In our study, the 0.1 M NaOH treatment TABLE I. TENSILE PROPERTIES OF SILK DEGUMMED WITH BOILING of A. atlas cocoons resulted in removal of a material WATER portion corresponding to nearly 20 % of their weight. On Silk E (MPa) u u (MPa) the other hand, SDS-PAGE analysis showed that much B. mori 685452.50 0.090.019 7261.00 lower amount of protein material was released by using A. atlas 29863.42 0.170.020 5114.69 this NaOH concentration from the A. atlas cocoons in comparison to the B. mori ones. Even water treatment of TABLE II. TENSILE PROPERTIES OF SILK DEGUMMED WITH 0.01M NAOH FOR B. MORI AND 0.1M NAOH FOR A. ATLAS B. mori cocoons liberated higher portion of material compared to that released from A. atlas cocoons by using Silk E (MPa) u u (MPa) the 0.1 M NaOH. Scanning electron microscopy analysis B. mori 3110645.17 0.080.012 23017.07 revealed that 0.01 M NaOH treatment of A. atlas was A. atlas 60436.65 0.170.012 10113.28 sufficient for removing of most extrafibrillar material,

however, fibers were still strongly tethered together and it was hard to reel them at dry or semidry conditions (not shown). Complete fibers releasing from A. atlas cocoons was obtained during treatment with 0.1 M NaOH; however, these fibers had similar morphology as the fibers after 0.01 M NaOH treatment as shown by SEM analysis (Fig. 3).These data indicates that efficient degumming by 0.1 M NaOH did not result only in removing of sericin layer but it acted likely also by another mechanism. It is known that tanned silks contain compounds which could be responsible for crosslinking Figure 4. Tensile analysis. Tensile strength of B. mori was found higher than A. atlas for both degumming protocol. For both silk fibers, alkali of proteins [21], [22]. We hypothesize that these tanning treatment yield better tensile strength fibers than that with boiling water. agents are present predominantly on the surface of fibers because cocoons homogenization (with Dounce homogenizer) yielded considerably lighter material (not shown). This is explainable by a pale color the interior of the fibers. As degumming by using 0.1 M NaOH turns the solution into brownish (tanned) color, we hypothesize that hydrolysis and/or release of tanned protein from the fiber surface occurred. Efficient degumming of silk fibers of related members from Saturniidae family by using 0.5 % Na2CO3 and 10% ethylenediamine was described previously [23]. Similar protocol was also used for A. atlas [24]. Our data show that A. atlas fibers are highly resistant to chemical treatment such as strong alkali conditions, which is in agreement with similar observation for A. pernyi. In this case, no significant change of silk fiber morphology and weight was observed after 1 M NaOH treatment for 24 h at room temperature [25]. Figure 5. Cell growth on B. mori and A. atlas fibers. The B. mori and A. The results of tensile test indicated that the B. mori atlas fibers were obtained by degumming of cocoons by using 0.01 M fiber has higher tensile strength compared to A. atlas. and 0.1 M NaOH, respectively. U2OS cells were allowed to attach to Similar result was published by Poza et al., 2002 who the fibers and cultivated for six days. After cell fixation, nuclei were used boiling water for degumming. However, our stained (using DAPI) and the material was analyzed by fluorescence and light microscopy. protocol using alkali treatment resulted in better tensile strength compared to boiling water treatment. This Natural silks represent material not only for textile applies for materials isolated from both silkmoths. industry but also potentially new material for broad bio- Degumming the cocoon by boiling water indicate that engineering applications. Our data show that mutual the adhesive proteins coating the fiber still remain on the stabilization forces between silk fibers differ in A. atlas silk surface especially form A. atlas. This is the reason cocoon compared to those in cocoon of the best described for different cross sectional diameters of the fibers [19]. natural silk moth B. mori. Here we show that NaOH can The differences in cross sections of the fibers create stress

riser on their surface during determination of tensile high molecular mass elementary unit consisting of H-chain, L- mode and create local escalation of stress that makes the chain, and P25, with a 6:6:1 molar ratio,” J. Biol Chem, vol. 275, pp. 40517-40528, 2000. fibers susceptible to break. This is in contrast to a smooth [8] J. Magoshi, Y. Magoshi, S. J. Nakamura, and S. J. Crystallization, surface deprived of adhesive protein coating as obtained “Liquid crystal, and fiber formation of silk fibroin,” Appl Polym by alkali treatment of both B. mori and A. Atlas cocoons. Symp, vol. 41, pp. 187-204, 1985. Despite different composition, primary structures and [9] M. Mondal and K. Trivedy, “The silk proteins, sericin and fibroin in silkworm Bombyx mori, Linn—a review,” Caspian J. Environ physical properties of fibroins from B. mori and members Sci, vol. 5, pp. 63–76, 2007. of Saturniidae [14], both types of fibroin could be used as [10] D. N. Rockwood, R. C. Preda, T. Yucel, X. Wang, M. L. Lovett, a matrix for cultivation of mammalian cells. Growth of and D. L. Kaplan, “Materials fabrication from Bombyx mori silk broad range of mammalian cells on the fibroin unwoven fibroin,” Nat Protoc, vol. 6, pp. 1612-1631, 2011 [11] C. Acharya, S. K. Ghosh, and S. C. Kundu, “Silk fibroin film from nets was described for B. mori [26] and also for fibers non-mulberry tropical tasar silkworms: A novel substrate for in isolated from A. atlas cocoons [24]. vitro fibroblast culture,” Acta Biomater, vol. 5, pp. 429–437, 2009. [12] C. Patra, S. Talukdar, T. Novoyatleva, S. R. Velagala, C. Mühlfeld, and B. Kundu, “Silk protein fibroin from antheraea mylitta for IV. CONCLUSION cardiac tissue engineering,” Biomaterials, vol. 33, pp. 2673-2680, In summary, we demonstrate here that NaOH could be 2012. [13] A. Datta, A. K. Ghosh, and S. C. Kundu, “Differential expression used as an efficient, cheap and environmentally safe of the fibroin gene in developmental stages of silkworm, antheraea degumming agent for easy releasing of A. atlas fibers. mylitta (saturniidae),” Comp Biochem Physiol B Biochem Mol This procedure requires stringent conditions represented Biol , vol. 129, pp. 197-204, 2001. by concentrations of 0.1 M NaOH for efficient [14] C. J. Fu, D. Porter, X. Chen, F. Vollrath, and Z. Shao, “Understanding the mechanical properties of antheraea pernyi degumming of A. atlas fibers probably due to presence of Silk—from primary structure to condensed structure of the the protein crosslink compounds. We show here that protein,” Adv Funct Mater, vol. 21, pp. 729–737, 2011. degummed fibers of A. atlas could be used for cultivation [15] M. Ho, H. Wang, and L. Kin-tak, “Effect of degumming time on of U2OS cells; human osteosarcoma cell line. This may silkworm silk fibre for biodegradable polymer composites,” Appl Surf Sci, vol. 258, pp. 3948-3955, 2012. be relevant for application of silk fibers for regenerative [16] P. Jiang, H. F. Liu, C. H. Wang, L. Z. Wu, J. G. Huang, and C. medicine. Guo, “Tensile behavior and morphology of differently degummed silkworm (Bombyx mori) cocoon silk fibres,” Mater Lett, vol. 60, pp. 919-925, 2006. ACKNOWLEDGMENT [17] G. V. Guinea, M. Elices, J. I. Real, S. Guiterrez, and J. P. Riguerio, This research was initiated by program of Scheme for “Reproducibility of the tensile properties of spider (Argiope trifasciata) silk obtained by forced silking,” J. Exp Zool, vol. 303A, Academic Mobility and Exchange sponsored by pp. 37-44, 2005. Directorate General of Higher Education of The Republic [18] J. P. Rigueiro, M. Elices, J. Llorca, and C. Viney, “Tensile of Indonesia and continued trough Scheme of properties of Attacus atlas silk submerged in liquid media ,” J. International research collaboration (Penelitian Appl Polym Sci., vol. 82, pp. 53-62, 2001. [19] P. Poza, J. P. Rigueiro, M. Elices, and J. Llorca, “Fractographic Kerjasama Luar Negri) of PNBP Udayana University. analysis of silkworm and spider silk,” Eng. Fract Mech, vol. 69, This work also supported by Czech Science Foundation pp. 1035-1048, 2002. project P108/12/G108 trough Institute of Chemical [20] T. Gheysens, A. Collins, S. Raina, F. Vollrath, and D. P. Knight, Technology of Prague. Special thank are addressed to Jan “Demineralization enables reeling of wild silkmoth cocoons,” Biomacromolecules, vol. 12, pp. 2257-2266, 2011. Lipov and Lenka Strnadová both from Department of [21] P. C. J. Brunet and B. C. Coles, “Tanned silks,” Proc. R Soc. Lond Biochemistry and Microbiology, Institute of Chemical B Biol. Sci. , vol. 187, pp.133–170, 1974. Technology, Prague for their permission and helpful [22] D. J. Raven, C. Earland, and M. Little, “Occurrence of dityrosine assistance during stay in the laboratory and also for Dr. in Tussah silk fibroin and keratin,” Biochim Biophys Acta, vol. 251, pp. 96-99, 1971. Josef Nahlik form the Department of Solid State [23] N. Reddy and Y. Yang, “Morphology and tensile properties of silk Engineering for electron microscopic analysis. fibers produced by uncommon saturniidae,” Int J. Biol. Macromol vol. 46, pp. 419–424, 2010. REFERENCES [24] N. Reddy, Y. Zhao, and Y. Yang, “Structure and properties of cocoons and silk fibers produced by Attacus atlas,” J. Polym [1] J. G. Hardy and T. R. Scheibel, “Composite materials based on Environ, vol. 21, pp. 16–23, 2013. silk proteins,” Prog Polym Sci, vol. 35, pp. 1093–115, 2010. [25] Y. Kawahara and M. Shioya, “Mechanical properties of tussah silk [2] X. Zhang, M. G. Reagan, and D. L. Kaplan, “Electrospun silk fibers treated with methacrylamide,” J. Appl. Polym Sci., vol. 65, biomaterial scaffolds for regenerative medicine,” Adv Drug pp. 2051-2057, 1997. Deliver Rev; vol. 61, pp. 988–1006, 2009. [26] R. E. Ungera, M. Wolfa, K. Petersa, A. Mottab, C. Migliaresib, [3] Y. Wang, H. J. Kim, G. Vunjak-Novakovic, and D. L. Kaplan, and C. J. Kirkpatricka, “Growth of human cells on a non-woven “Stem cell-based tissue engineering with silk biomaterials,” silk fibroin net: a potential for use in tissue engineering,” Biomaterials, vol. 27, pp. 6064-6082, 2006. Biomaterials, vol. 25, pp. 1069–1075, 2004. [4] B. Panilaitis, G. H. Altman, J. Chen, H. J. Jin, V. Karageorgiou, and D. L. Kaplan, “Macrophage responses to silk,” Biomaterials, Tjokorda Gde Tirta Nindhia Was born in vol. 24, pp. 3079–3085, 2003. Denpasar, Bali, Indonesia on January 16th, [5] L. Meinel, S. Hofmann, V. Karageorgiou, C. Kirker-Head, J. 1972. Received Doctor Degree in Mechanical McCool, G. Gronowicz, et al., “The inflammatory responses to Engineering from Gadjah Mada University silk films in vitro and in vivo,” Biomaterials, vol. 26, pp. 147–55, (UGM) Yogyakarta, Indonesia on August 2005. 2003, with major field of study was Material [6] F. Vollrath and D. Porter, “Silks as ancient models for modern Engineering. polymers,” Polymer, vol. 50, pp. 5623–5632, 2009 He participated in various international [7] S. Inoue, K. Tanaka, F. Arisaka, S. Kimura, K. Ohtomo, and S. research collaborations such as with Muroran Mizuno, “Silk fibroin of Bombyx mori is secreted, assembling a Institute of Technology Japan (2004),

Toyohashi University of Technology Japan (2006), Leoben Mining Zdenek Knejzlik is senior researcher at Faculty of Food and University Austria (2008-2009), Technical University of Vienna Austria Biochemical Technology, Technicka 5, 166 28 Prague Czech Republic (2010) and Recently with Institute Chemical technology of Prague I Wayan Surata was born in Nusa Penida, Bali, Indonesia on July 5, Czech Republic (2012-now). His current job is as Full Professor in the 1958. Received Doctor Degree in the field of Ergonomic from Udayana field of Material Engineering at Department of Mechanical Engineering, University in 2011. His research interest very much related in process of Engineering Faculty, Udayana University, Jimbaran, Bali, Indonesia. manufacture. His Current job is researcher and lecturer at Department of His research interest covers subjects such as, biomaterial, waste recycle, Mechanical Engineering, Engineering Faculty, Udayana University, failure analyses, ceramic, metallurgy, composite, renewable energy, and Jimbaran, Bali, Indonesia. environmental friendly manufacturing. Tjokorda Sari Nindhia is Doctor of Veterinary Medicine (DVM) at Tomas Ruml is full professor and dean of the Faculty of Food and faculty of Veterinary Medicine Udayana University, Bali, Indonesia Biochemical Technology, Technicka 5, 166 28 Prague Czech Republic since 1999. Finishing her master degree at biotechnology and biomolecular at postgraduate studies Udayana University in 2010