Professional Documents
Culture Documents
net/publication/305865680
CITATIONS READS
5 1,925
5 authors, including:
Some of the authors of this publication are also working on these related projects:
Potential of Comamonas acidovorans for industrial and remediation application View project
All content following this page was uploaded by Ankit V Patel on 06 August 2016.
ABSTRACT:
Bio-based plastics are novel approaches which are found to be good alternative for petroleum
based plastics. Protein, extracted from defatted cake is better natural polymer in comparison
with petroleum based polymer because of it is a renewable resources, easily available,
economically cheaper and environmentally friendly. Polymeric blends of rapeseed defatted cake
isolated protein and PVA have been the major ingredients of bio-plastics. Various conditions
were optimized for extraction of protein from rapeseed defatted cake. Bio-plastic sheets were
formulated by compression moulding of protein isolates, PVA, glycerol, silicon oil and calcium
carbonate. The concentration of filler was optimized to increase the strength and efficiency of
bioplastics sheet. Biodegradation of bioplastic was done using bacteria cultures in the soil and
liquid medium to understand the disposal possibilities of bioplastic in the laboratory along with
natural conditions. Degradation of rapeseed protein based bioplastic was found 57% and 74%
in the soil and liquid medium respectively.
INTRODUCTION:
The utilization of Petro-based plastic has become a day to day need of every human being. The
amount of plastic that is discarded every year end up in landfills and water which has been a
threat for the aquatic as well as terrestrial living beings [1]. The need of population cannot be
decreased and hence, a need to develop new materials to substitute synthetic polymers has
become an important challenge nowadays [2]. Polymers from renewable resources have been
attracting ever-increasing attention of researchers over the past two decades, predominantly for
two reasons: first being environmental concerns and the second being the realization that our
petroleum resources are finite [3, 4]. Plastic waste disposal is a huge eco-technological problem
1
Department of Industrial Chemistry, Institute of Science and Technology for Advanced studies and Research
(ISTAR), Vallabh Vidyanagar, Anand, Gujarat-388 120, India.
2
Department of Microbiology, Natubhai V. Patel college of Pure and Applied Sciences, Vallabh Vidyanagar,
Anand, Gujarat-388 120, India.
*Responding Author
© 2016 I licensee IJCST. This is an Open Access Research distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution,
and reproduction in any Medium, provided the original work is properly cited.
FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES
and one of the approaches to solve this problem is the development of biodegradable plastic-bio
plastic [5]. Biopolymers from agricultural sources are becoming an interesting alternative not
only as biodegradable films suitable for food packaging, but also as plastic stuffs, which require
improved mechanical properties [6, 7]. Proteins [8, 9], starch [10, 11] and polysaccharides [12]
have been used as polymer sources for many years [13, 14].
Many researchers have made an attempt to make protein based bioplastic as proteins are
occurring naturally [15]. Protein and synthetic petroleum based polymer shared some vital
characteristics such as a synthetic polymer consists of identical polymer, covalently bonded in a
long chain, while protein are composed of repeating units of different amino acids which is
called as polypeptide chain[16]. Protein can be easily extracted from various renewable sources
viz. de-oiled cake, maize gluten, etc. Protein based bioplastic such as edible film or articles have
been made by casting or compression molding is reported in the literature [17, 18]. Different
bacterial and fungal cultures were evaluated for the degradation of bioplastic as a sole source of
carbon source. Bacterial cultures such as Rhodococcussp., Enterobactordissolvans, Bacillus
subtilis, and Pseudomonas aeruginosa showed efficient degradation within 2 to 9 months[19].
Comamonasacidovorans MTCC 3364 is an efficient degrader of different azo dyes and heavy
metal [20]. Extracts of bioplastic offered a more advantages like increased soil fertility and low
accumulation of plastic materials in the ecosystem.
In the present study, the extraction of protein was obtained from rapeseed defatted cake [21].
Defatted cake is waste, which is generated from oil seed after extracting oil from oil seed. The
extracted protein was blended with filler, binder, lubricants and plasticizer, which are also
biodegradable and each have its specific explanation to use in the formulation of the sheet. Filler
is use in composition of sheet due to provide better flow-ability along with to decrease product
cost. A plasticizer is used because of its imparted flexibility to the bioplastic sheet [22]. The
composition might contain minor but effective amount of a lubricant to provide a lubricating
effect to the article into the mold and release article easily from the mold. The concentration of
protein, plasticizer, binder and filler was optimized for the fabrication of rapeseed protein based
bioplastic.
Mechanical, surface and microstructure characterization was done using a universal testing
machine (UTM), impact tester, field emission gun scanning electron microscopy (FEG-SEM).
Biodegradation of bioplastic was done under natural as well as a laboratory conditions in the soil
[23]. Defined liquid medium used for faster and improved degradation study of bioplastic in
laboratory condition was also evaluated. The microstructure of the degraded sheets was also
evaluated under scanning electron microscopy.
3. Extraction of protein from Rapeseed Defatted cake (DFC) and estimation of protein
Protein was extracted from rapeseed DFC in alkaline condition. Rapeseed DFC was dissolved in
sodium hydroxide solution (0.1M to 2M), keeping the ratio of 1:20 (w/v) (DFC: Sodium
hydroxide). The solution was heated up to 50°C in the time frame of 1 to 5 h, at desired stirring
speed for dissolution of protein from defatted cake. The extract containing protein solution was
filtered using linen cloth to remove the residue. Precipitation of protein was carried out by
adding 0.1N hydrochloric acid drop wise, till iso-electric point was reached. The resultant protein
was collected using centrifuge and was dried at 45°C in hot air oven. The dry protein powder
was use for estimation of protein and preparation of bio-plastics sheet [25].
The Folin-Lowry protein assay is a biochemical assay for determining the concentration of
protein. Different aliquots of standard Bovine Serum Albumin (BSA, 200 µg% (w/v)) were taken
in different test tubes in the range of 0.2 to 1.0 ml. Five ml of Lowry’s reagent (alkaline copper
sulphate) was added into all the tubes and incubated for 10min at room temperature. After
incubation, 0.5 ml of 1:2 dilute Folin’s reagent was added in each test tube and allowed to react
for 30min at room temperature in dark place. The optical density of samples was measured at
750 nm using spectrophotometer. The plot of optical density v/s concentration was constructed
and the protein content of unknown samples was measured [26].
min. The prepared bio-plastic sheet was collected after mould was cooled.The mechanical
properties, thermal property and surface morphology of the resultant bio-plastic sheets were
measured.
5. Mechanical properties
Mechanical properties of the resultant bio-plastic sheet were evaluated depending on its
application using ASTM standard.
5.1 Tensile strength and % Elongation
The tensile strength of the resultant bio-plastic sheets were determined according to the ASTM
D-638) using universal testing machine (Shimadzu AG-100kN, Japan) [27]. A dumb-bell shaped
specimen was used for the tensile strength and % elongation. Initial grip separation was set at 20
mm; while cross-head speed was set at 50 mm/min. Thickness and width of the samples were
measured using calipermicrometer. Tensile strength was calculated by dividing maximum load
developed during the test by initial sheet cross sectional area. % elongation at break was
calculated by dividing sheet extension at the moment of rupture with an initial length of the sheet
and multiplying it by 100.
The formula of the tensile strength and % elongation was described as follows:
3𝑃𝑃𝑃𝑃 𝑃𝑃𝑃𝑃³
Flexural strength =2𝑏𝑏𝑏𝑏 ² Flexural modulus = 4𝑏𝑏𝑏𝑏 ³𝑦𝑦
Where, P = three point load, L = span length, b = breath of the sample, d = depth of the sample,
and y = difference between initial and final deflection
was consists of 12 flasks for each microorganisms, 6 flasks was biotic (with microorganisms)
and another 6 flasks was abiotic control (without microorganisms). Individual samples were
extracted after every 6 days, and analysed periodically for weight loss (%). One ml of the sample
was taken out for the optical density was measured determined at 600 nm. Liquid culture was
filtered using Whatman filter paper; degraded bioplastic sheet was removed and kept in the hot
air oven for drying at 50°C. This filtrate was further proceeding to centrifugation at 7500 rpm for
10 min. % weight loss was measured by the same method as above.
Table 2: Weight of extracted crude protein from rapeseed defatted cake and % protein present
in the crude protein
Normality of NaOH
Time 0.1M 0.5M 1M 2M
(h) g % g % g % g %
1 3.10 81.00 4.39 89.93 3.84 80.49 3.28 76.04
2 3.19 86.45 4.01 91.48 3.29 81.93 2.97 79.73
3 4.29 92.47 4.69 94.35 4.24 89.25 3.90 84.93
4 3.90 80.93 4.38 85.34 3.93 74.48 3.04 69.82
5 3.52 75.84 3.73 79.45 3.38 71.72 2.73 64.38
Tensile strength is an essential mechanical property that express maximum stress which develops
in the sheet during tensile testing and elongation is the greatest change in the length of the test
specimen before breaking. As the percentage of calcium carbonate increases, the tensile strength
of the sheet increased gradually while % elongation of the sheet decreased. An extracted protein
consists of polar and non-polar side chains. There are strong intra and inter molecular
interactions with other compounds, such as hydrogen bonding, hydrophobic interactions, etc.
which are contribute to tensile strength. As the concentration of filler increases in the
composition of sheet, percentage of plasticizer in overall mixture decreases because of that %
elongation of sheet decreases. Along with an increase in tensile strength impact strength also
increases. Among the three bioplastics sheets of rapeseed protein, the sheet had 60% filler
showed highest tensile strength, while it has lowest percentage elongation. Wang et al. 1999
revealed that protein/starch based bio-plastic had 3.05-18.35kg f/cm2 tensile strength, whereas
bio-plastics sheets using rapeseed protein had 7.87-22.64kg f/cm2tensile strength [32].The results
of flexural strength showed that as concentration of filler increases; flexural strength and flexural
modulus both were increased.
From this study it can be observed the strength of the bioplastic increase with increase in
concentration of calcium carbonate and along with it, calcium carbonate is a natural and non-
toxic filler it could help in degradation of the bioplastics without any harmful effect to the
environment.
35
30
25
20
15
10
5
0
Farmland soil Compost soil Sandy soil
Soil type
Figure 1: Rapeseed protein based bioplastic sheets degradation under different soil natural
conditions
70
60
Weight loss (%)
50
40
30
20
10
0
6 12 18 24 30 36 42 48
Days
release of primary and secondary metabolites by the organisms and degradation of bioplastic
[41].
80 2
60 1.5
50
40 1 Weight loss of abiotic control (%)
30
Weight loss using organism (%)
20 0.5
10 Optical density (600 nm)
0 0
6 12 18 24 30 36
Days
80 2.5
2
60
50 1.5
40 Weight loss of abiotic control (%)
30 1
20 Weight loss using organism (%)
0.5
10 Optical density (600 nm)
0 0
6 12 18 24 30 36
days
80
70 2
Weight loss (%)
60
1.5
50
40 Weight loss of abiotic control (%)
1
30 Weight loss using organism (%)
20 0.5 Optical density (600 nm)
10
0 0
6 12 18 24 30 36
days
CONCLUSION
The proposed method for extraction of protein from rapeseed de-oiled cake is simpler and gives
better quality of protein with maximum yield. 0.5M NaOH, 3 h time, 1:20 (W/V) rapeseed
DFC:NaOH at 50°C temperature are the optimum conditions for protein extraction from
rapeseed DFC. A protein extracted from rapeseed DFC is of 94.35% purity. A protein isolated
using above technique was blended with PVA, glycerol, and calcium carbonate for formulating a
bio-plastic sheet. The mechanical properties of sheet vary with the change in the amount calcium
carbonate filler. As the percentage of calcium carbonate in sheet increases, the tensile strength
and the impact strength of the resultant bioplastic sheet also increases. The bioplastic sheet
prepared using 60% calcium carbonate showed good mechanical properties compared to other
two sheets. The degradation bioplastic sheet in natural condition soil was about 44%, whereas
consortium depicted highest degradation about 57% and 74% in laboratory condition in the soil
and in liquid medium respectively. Results of biodegradation shows that, there is no inhibition
against normal soil and water flora which depicted the disposal of the bioplastic sheets,it is not
generating any type of pollution in the environment. Rapeseed protein based bioplastic sheets
can be used for making disposable articles such as spoon, cups, and light weight utensils.
Acknowledgement
The authors want to acknowledge Sophisticated Instrumentation Centre for Applied Research
and Testing (SICART), V.V. Nagar, Gujarat, for providing the necessary instrumentation
facilities. The authors also acknowledge the Microbial Type Culture Collections and Gene Bank,
Institute of Microbial Technology, (Chandigarh, India) for providing the bacterial culture.
REFERENCES
[1] Barnes, D. K. A.;Galgani, F.; Thompson, R. C.;Barlaz, M. Phil. Trans. R. Soc. 2009, 364,
1985–1998.
[2] Müller, C.; Townsend, K.;Matschullat, J. Sci total environ. 2012, 416, 464-467.
DOI.10.1016/j.scitotenv.2011.10.069.
[3] Yu, L.; Chen, L. Biodegradable polymer blends and composites from renewable resources.
2009:1-15. john Wiley and sons, inc.
[4] Romero, A.; Guerrero, A.; Felix, M.; Martin-alfonso, J.E. J food eng. 2014, 125, 7-16.
[5] Flieger, M.;Kantorova, M.;Prell, A.;Rezanka, T.;Votrubu, J. Folia microbial. 2003, 48(1), 27-
44.
[6] Scheller, j.; Conrad, U. Curropin plant boil. 2005, 8, 188-196.
[7] Gómez-Martínez, D.;Partal, P.;Martínez, I.; Gallegos, C. Bioresource technol. 2009, 100,
1828–1832.
[8] Martínez, I.;Partal, P.;García-Morales, M.; Guerrero, A.; Gallegos, C. J food eng. 2013,
117(2), 247-254.DOI: 10.1016/j.jfoodeng.2013.02.014
[9] Zhang, J.;Mungara, P.; Jane, J. J of Polymer. 2001, 42, 2569-2578.
[10] González-Gutiérrez, J.;Partal, P.;García-Morales, M.; Gallegos, C. CarbohydPolym. 2011,
84(1), 308-315. DOI./10.1016/j.carbpol.2010.11.040.
[11] Kaewkannetra, P.;Soonthornchiya, J.;Moonamart, S. Jbiotechnol. 2010, 150, 385.
DOI:10.1016/j.jbiotec.2010.09.482.
[12] Zárate-Ramírez, L. S.; Romero, A.;Bengoechea, C.;Partal, P.; Guerrero, A. CarbohydPolym.
2014, 112, 24-31.DOI:10.1016/j.carbpol.2014.05.055.
[13] Ahn, H. K.; Huda, M. S.; Smith, M. C.;Mulbry, W.; Schmidt, W.F.; Reeves, J. B.
Bioresource technol. 2011, 102(7), 4930-4933. DOI:10.1016/j.biortech.2011.01.042.
[14] Hewage, S.;Sashiprabha, M.;Vithanarachchi. J.Natn.sci 2009, 37(1), 53-59.
[15] Félix, M.; Martín-Alfonso, J. E.; Romero, A.; Guerrero, A. Jfood eng. 2014, 125, 7-
16.DOI:10.1016/j.jfoodeng.2013.10.018.
[16] Casparus, J.; Verbeek, R.; Lisa, E.; Van, D. B. Macromole mater eng. 2010, 295, 10–21.
DOI: 10.1002/mame.200900167.
[17] Guerreroa, P.; Stefani, P. M.;Ruseckaite, R. A.; de la Caba, K. J food eng. 2011, 05, 65–72.
[18] Wang, S.; Plano, T. U.S.Pat.No. 5,922,379.Biodegradable protein/starch based
thermoplastic composition.
[19] Patel, D.;Toliwal, S. D.; Patel, J. V.;Gupte, A.; Patel, Y. Polym-plast technol. 2011, 50, 332-
338. DOI: 10.1080/03602559.2010.531995.
[20] Rudakiya, D.;Pawar, K. IOSR-JESTFT. 2013, 5(5), 26-32. DOI:10.9790/2402-0552632.
[21] Gómez-Martínez, D.;Partal, P.;Martínez, I.; Gallegos, C. Ind cropprod. 2013, 43, 704-710.
DOI:10.1016/j.indcrop.2012.08.007.
[22] Song, Y.; Zheng, Q. Bioresource technol. 2008, 99(16), 7665-
7671.DOI:10.1016/j.biortech.2008.01.075.
[23] Tokiwa, Y.;Calabia, B. P.;Ugwu, C. U.;Aiba, S. International Journal of Molecular Science
2009, 10, 3722-3742. DOI:10.3390/ijms10093722.
[24] BIS: 548 (Part- I) (1964), Method of sampling and tests for oils and fats, (Bureau of Indian
standard, (New Delhi).
[25] Toliwal, S. D.; Patel, K. J sciind res india. 2007, 66, 385-387.
[26] Lowry, O. H.;Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J Biol Chem.1951, 193, 265-
275.
[27] ASTM standard D638-10, 2008. Standard Test Method for Tensile Properties of Plastics
ASTM International, Conshohocken, PA, 2008. DOI: 10.1520/D0638-10. www.astm.org
[28] ASTM D256-10, Standard Test Methods for Determining the Izod Pendulum Impact
Resistance of Plastics. DOI: 10.1520/D0256-10. www.astm.org
[29] ASTM D790-10, Standard Test Methods for Flexural Properties of Unreinforced and
Reinforced Plastics and Electrical Insulating Materials. DOI: 10.1520/D0790-10.
www.astm.org
[30] Londhe, S. V.; Joshi, M. S.;Bhosale, A. A.; Kale, S. B. 2011, 2(3), 1175-1177.
[31] Patel, D.;Toliwal, S, D.; Patel, J. V. Polym-plast technol. 2012, 51, 238-242.
[32] Wang, S.; Plano, T. U.S.Pat.No. 5,922,379. Biodegradable protein/starch based
thermoplastic composition.
[33] Domenek, S.;Feuilloey, P.;Gratraud, J.; Morel, M. H.;Guilbert, S. Chemosphere. 2004, 54,
551-559. DOI: 10.1016/S0045-6535(03)00760-4.
[34] Yang, H. S.; Yoon, J. S.; Kim, M. N. Polymdegradstabil. 2004, 84(3), 411-7.
[35] Kim, H. S.; Kim, H. J.; Lee, J. W.; Choi, I. G. Polymdegradstabil. 2006, 91, 1117-1127.
DOI:10.1016/j.polymdegradstab.2005.07.002
[36] González, A.;Strumia, M. C.;Igarzabal, C. I. A. J food eng. 2011, 106(4), 331-
338.DOI:10.1016/j.jfoodeng.2011.05.030.
[37] Kathiresan, K. Rev Biol Trop. 2003, 73, 363-75.
[38] Singh, B.; Sharma, N. Polymdegradstabil. 2008, 93, 561-584.
[39] Yang, H. S.; Yoon, J. S.; Kim, M. N. Polymdegradstabil. 2005, 87, 131.
[40] Sivan, A. Curropin biotech. 2011, 22, 422–426. DOI 10.1016/j.copbio.2011.01.013.
[41] Patel, Y.; Mehta, C.;Gupte, A. Intbiodeterbiodegr. 2012, 75, 187-193.
[42] Lopez-Liorca, L. V.;Valiente, M. F. C. Micron. 1993, 24(5), 457-63.