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FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS

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International Journal of Chemical Sciences and Technology
ISSN: 2455-3263
Volume 1, Issue 3, DIP: 18.04.001/20160104
www.ijcst.redmac.in | July-August, 2016

FABRICATION OF BIO-PLASTICS FROM PROTEIN

ISOLATES AND ITS BIODEGRADATION STUDIES

Ankit V. Patel 1, Tirth M. Panchal1, Darshan Rudakiya 2,

Akshaya Gupte2, Jigar V. Patel1*

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.

KEYWORDS: Bio-plastic, defatted cake, biodegradation study, mechanical properties, surface

morphology, compression moulding.

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.

© International Journal of Chemical Sciences and Technology | 2

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

MATERIALS AND METHOD:

1. Raw materials and Chemicals
Rapeseed Defatted cake were procured from Sunrise cattle feed Pvt. Ltd. (Anand, Gujarat,
India). Polyvinyl alcohol (PVA), glycerol, silicon oil were purchased from Sigma (St. Louis,
U.S.A). Sodium hydroxide, hydrochloric acid, bovine serum albumin (BSA) and calcium
carbonate was purchased from HI-media (Mumbai, India).

2. Analysis of Defatted cake

Defatted cakes of rapeseed were analysed for protein content, moisture content, fibre content and
oil content by standard methods. Proteins were estimated in these defatted cakes from the
nitrogen content by the kjeldahl method. The result of analysis are tabulated in table 1 [24].

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].

4. Formulation and fabrication of bio-plastic sheets

To prepare a bio-plastic sheet Rapeseed protein isolates (RPI), poly vinyl alcohol (PVA) and
other additives were mixed thoroughly in a two roll mill. General composition for protein based
bio-plastic sheet was RPI (41%), PVA (41%), and glycerol (18%). Silicon oil (0.5%) and
calcium carbonate (CaCO3) were added as a lubricant and filler respectively. Various
concentrations (40%, 50% and 60%) of the CaCO3 filler was added by total weight of the
respective sheets and this mixture was blended using two roll mill. Also prepared bioplastic
sheets without CaCO3 considered as a control. In two roll mill, mixture was blended for 20 min
at 100°C, after blending it was palletized. These pellets were compressed in compression
moulding at 140°C for 2 h without applying pressure after that 40 kg/cm2 pressure applied for 10

© International Journal of Chemical Sciences and Technology | 3

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

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:

Load recorded at the break

Tensile strength = Cross sectional area (b×d)

Change in length ×100

% elongation = Original length

Where, b = breath of the sample

d = depth of the sample

5.2 Impact strength

Impact strength is a method for determining the impact resistance of the sheet. It is a
standardized high strain rate test which determines the amount of energy absorbed by a material
during fracture. Impact strength of the resultant bio-plastic sheets were determined according to
the ASTM D-256 using impact tester (Model DG-UB2, Toyo seikiseisaku-sho Japan) [28].

5.3 Flexural strength and modulus

The flexural strength and flexural modulus of the resultant bio-plastic sheet were measured by
three point bending method. Flexural strength represents the highest stress experienced within
the material at its moment of rapture. The testing of the resultant bio-plastic sheet was done
according to the ASTM D-790 using a universal testing machine (Shimadzu AG-100kN, Japan)
[29].The specimen of rectangular cross section (3.12 mm × 15.6 mm × 105 mm) which behaves
as a beam tested in flexure. The cross head speed was 5 mm/min. The formula is used for
calculating flexural strength and modulus as follows:

© International Journal of Chemical Sciences and Technology | 4

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

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

6 Degradation studies of bio-plastic sheets

Biodegradation studies of the fabricated bioplastic sheet were performed using various methods
viz. natural condition, laboratory condition in soil and liquid medium.

6.1 Degradation study of bio-plastic under natural condition

Degradation of the protein based bioplastic sheets was carried at under natural condition such as
farm land soil, compost soil and sandy soil. Bioplastic sheets (2 g) were inoculated at the depth
of the 10 cm under the respective soil. For compost soil degradation, 100 g of compost (25 cm x
25 cm x 3 cm) was dumped at the depth of 5 cm of farmland soil. The moisture ratio of the soil
was maintained approx. 50-60% and distilled water was sprayed on the soil. Biodegradability of
the bioplastic was evaluated by measuring % weight loss of the sheets after incubation of the 48
days. Bioplastic sheets were extracted, washed with distilled water for removing the unwanted
soil particles and dried in hot air oven at 50°C until the constant weight of bioplastic sheet was
obtained. % weight loss was measured using the following formula:
(Wi −Wf )
% weight loss = Wi
× 100

Where, Wi = Initial weight of bioplastic sheet, Wf = Final weight of bioplastic sheet

6.2 Degradation study of bioplastic under laboratory condition in soil

Farmland soil was used for the degradation analysis of bioplastic sheets under laboratory
condition. It was sterilized (15 lbs pressure) for 45 min at 121°C. Soil analysis was done to
understand the nutrient composition of the soil from GSFC (Vadodara, Gujarat). Soil analysis
results of the farmland soil was given as follows: C:N (0.43%), P (36 kg/Ac), K 108 kg/Ac, S
(22.30 mg/L), Cu (2.32 mg/L), Fe (62.60 mg/L), Mn (28.76 mg/L), and Zn (0.7 mg/L). Plastic
cups were taken for the degradation analysis. 1 g of bioplastic sheets was kept at the depth of the
5 cm of the cup and bacterial cultures (ComamonasacidovoransMTCC 3364, Bacillus
megaterium and consortium) were inoculated (10 ml, O.D. 1.00) around the bioplastic sheets.
The moisture ratio of the soil was maintained by spraying distilled water on soil (50-60% of
humidity). % weight loss was measured by the same method as above.

6.3 Degradation study of bio-plastic under liquid medium

Comamonasacidovorans MTCC 3364, Bacillusmegaterium and consortium of both bacteria were
taken for the liquid degradation medium. An experiment containing mineral salt medium (MSM)
was autoclaved at 121°C for 15 min, inoculated with bacterial cultures in individual flasks and
incubated at room temperature in static condition for 36 days. In experiment of biodegradation

© International Journal of Chemical Sciences and Technology | 5

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

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.

7 Field Emission Gun-Scanning Electron Microscopy

Air dried degraded bioplastic sheet was studied using field emission gun scanning electron
microscopy (FEG Nano Nova SEM 450, FEI Ltd., USA) under a high vacuum condition at 5.00
kV with 2000x magnification for detection of microstructure and surface morphology.

RESULTS AND DISCUSSION

Protein extracted from rapeseed defatted cake was blended with plasticizers, filler and other
additive to prepare a bio-plastic sheet. Protein concentration in rapeseed defatted cake was
45.68%and fat content was 1.59%. Fibre, ash, total carbohydrate and moisture content of
rapeseed DFC are shown in table 1. Bioplastic sheet was tested for its mechanical property,
biodegradability and its microstructures. Results of these studies are discussed as under.

Table 1: Analysis of Defatted cake

Protein Moisture Fibre Ash Total

Defatted Oil content
content content content content carbohydrate
cake (%)
(%) (%) (%) (%) (%)

Rapeseed 1.59 45.68 9.58 11.05 5.30 26.8

1 Effect of concentration of Sodium hydroxide on protein extraction

Protein was extracted from Defatted cake in alkaline conditions. The ratio of DFC: alkaline
solution was of 1: 20, and the extraction was carried out at 50°C. As shown in table 2, various
concentrations of sodium hydroxide were used for extraction of protein from rapeseed DFC. The
yield of protein extracted was decreased by increasing concentration of sodium hydroxide which
can be due to the more hydrolysis of protein. Londhe et al. also revealed same that the rate of
hydrolysis was increased at the high concentration of sodium hydroxide [30, 31]. 0.5M sodium
hydroxide was found to be the optimum concentration for extraction of protein from rapeseed
DFC respectively.

© International Journal of Chemical Sciences and Technology | 6

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

2 Effect of time on protein extraction

As shown in the results (table 2), by increasing the time for extraction, protein yield was
increased. Extended extraction depicted the hydrolysis of the protein which decreased the protein
yield. Optimised protein yield obtained in 3 h for rapeseed protein.

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

3 Effect of Calcium carbonate as filler on bio-plastic sheet’s mechanical properties

Mechanical properties like tensile strength, percentage elongation, flexural strength as well as
flexural modulus were determined using the Universal testing machine according to their ASTM
method. Effect of calcium carbonate, as filler, on the mechanical properties of bio-plastic sheet
are shown in table 3.

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-

© International Journal of Chemical Sciences and Technology | 7

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

toxic filler it could help in degradation of the bioplastics without any harmful effect to the
environment.

Table 3: Mechanical properties of rapeseed protein based bioplastic sheet

Percentage Tensile Tensile Flexural Flexural Percentage Impact
of Calcium strength modules strength modulus elongation strength
Carbonate (kg f/cm2) (kg f/cm2) (kg f/cm2) (kg f/cm2) (%) (J / cm)
40 15.92 31.93 9.94 105.83 18.95 0.585
50 19.14 37.59 15.46 213.54 14.48 0.619
60 22.64 42.63 18.53 539.87 10.69 0.838
*Ratio of Protein: PVA: Glycerol was kept unchanged as 41:41:18

4 Degradation study of bio-plastic under natural condition

The weight loss study clearly depicted that bioplastic sheets could be degraded easily in the soil
in natural condition. From the degradation studies it was observed that protein based bioplastic
sheet degraded the most in compost soil rather than farmland and sandy soil (fig. 3). Degraded
sheet observed in the compost soil and farmland soil was 44.18 ± 0.90% and 35.73 ± 1.10%
respectively [33-35]. The reason of degradation of sheet in compost soil seems to be its
enrichment of nutrients and micro as well as macro flora. Sandy soil could degrade the least
amount of bioplastic sheets (21.90 ± 0.5%) because of soil did not contain any high amount of
nutrients along with the major flora of micro-organisms [36].
50
45
40
Weight loss (%)

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

© International Journal of Chemical Sciences and Technology | 8

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

5. Degradation of bioplastic under laboratory condition in soil

Soil analysis of the farmland soil was given as follows: C:N (0.36%), P (31 kg/Ac), K98 kg/Ac,
S (22.30 mg/L),Cu (1.98 mg/L), Fe (9.90 mg/L), Mn (24.30 mg/L), and Zn (1.06 mg/L).
Laboratory condition gave a higher possibility of degradation by specific type of organisms and
organism’s efficacy about to degradation. Maximum degradation was done using consortium on
(57.43%) and Comamonasacidovorans MTCC 3364 (55.76%) on 48th day depicted in the fig. 4.
Approximately 15-18% degradation of bioplastic sheets was done on the 6th day which showed
that there was a higher degradation rate of bioplastic [37]. This result suggests that both
Comamonasacidovorans MTCC 3364and Bacillus megaterium penetrate, attack on bioplastic
surface and degrade the rapeseed based bioplastic.
Comamonas acidovorans MTCC 3364 Bacillus megaterium Consortium

70

60
Weight loss (%)

50

40

30

20

10

0
6 12 18 24 30 36 42 48

Days

Figure 2: Rapeseed protein based Bioplastic sheets degradation by different micro-organisms

in farmland soil under laboratory condition.

6 Degradation study of bio-plastic in liquid medium

Degradation of rapeseed protein based bioplastic study was shown in the figure 3, 4 and 5.
Maximum degradation was done by Consortium of ComamonasacidovoransMTCC 3364 and
Bacillus megaterium on 36th day and rapid degradation was observed on the 6th day was 24%.
From the results, it is shown that actual weight loss of bioplastics due to microorganism is
56.43% in consortium. Consortium shows synergetic effect on degradation of bioplastics.
Maximum bacterial growth was depicted in the Bacillus megateriumwas1.944 nm. Soluble
protein determination was also suggested the removal of protein from the bioplastic sheet. Patel
et al. revealed that 62.06% degradation was observed by using Pseudomonas aeruginosa on the
52thday of inoculation under static condition [19]. The degradation of bioplastic sheets and
polyethylene sheet was done by using the different bacteria by many researchers in which
degradation study was done from 2 to 9 months [38-40]. The carbon load of the bioplastic
extracts was gradually increased in all three bacterial systems; these were done because of

© International Journal of Chemical Sciences and Technology | 9

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

release of primary and secondary metabolites by the organisms and degradation of bioplastic
[41].
80 2

Optical density (600 nm)

70
Weight loss (%)

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

Figure 3: Degradation of rapeseed based bioplastic in liquid medium by

Comamonasacidovorans MTCC 3364

80 2.5

Opticaal density (600 nm)

70
Weight loss (%)

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

Figure 4: Degradation of rapeseed based bioplastic in liquid medium by Bacillus megaterium

90 2.5
Optical density (600 nm)

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

Figure 5: Degradation of rapeseed based bioplastic in liquid medium by Consortium

© International Journal of Chemical Sciences and Technology | 10

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

7. Field Emission Gun-Scanning Electron Microscopy

The microstructure of the degraded bioplastic sheet was evaluated by the field emission gun
electron microscopy. Bacterial cells degraded the surface of the rapeseed based bioplastic sheets
shown in Fig. 6. From the FEG-SEM images, it was seen that bacteria degraded the surface of
the bioplastic and degradation is gradually increased as time increased, it’s shown in Fig. 6 A-B.
Maximum rupture of the sheet was shown in the fig. 6 B on the 36th day of degradation [18].
Before, biodegradability of bioplastic sheet has smooth surface. After biodegradability of sheet
on 36th in the liquid medium, large holes on sheet surface was increased which clearly depicted
the degradation of bioplastic sheet was due to by microorganisms surface attacked. [35, 42].

Figure 6: FEG-Scanning Electron Micrograph of rapeseed degraded bioplastics sheet

extracted from the liquid medium by consorsium on the 12th day (A), 36th day (B).

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

© International Journal of Chemical Sciences and Technology | 11

 FABRICATION OF BIO-PLASTICS FROM PROTEIN ISOLATES AND ITS BIODEGRADATION STUDIES

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.

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