Eco-Friendly Preparation and Characterization of Bioplastic Films from Marine Fish Wastes

Synthetic plastics are highly hazardous contaminants; hence they must be replaced with alternatives. This study aimed to prepare corn starch-based bioplastics using sh scale through lm casting technique as an alternative to synthetic plastics. In this work, four types of bioplastic lms containing different percentages of sh-scale powder and corn starch were prepared. Physical and chemical properties such as texture, color, solubility in hot water, tensile strength, organic content, and morphology of all the four types of the synthesized biopolymer were analyzed. The mixture of sh-scale powder and corn-our powder in the ratio of 1: 3 yielded the best results. In the biodegradability test, degradation was noticed after 7 days of treatment with organic waste. The degradation was conrmed by surface changes in the morphology and the development of Aspergillus sp. The produced bioplastics were synthesized from eco-friendly, inexpensive, and natural materials. Thus, the present research has provided a viable alternative to synthetic plastics.


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The global sh production is witnessing an increasing trend, and the annual capture shery production exceeded 150 million tons (FAO, 2018). This huge production also involves the generation of shery byproducts. Parts of shes such as head, skin, scales, skeletons, viscera, etc., amounting to 25 -75 % (Olsen et al., 2014), were discarded as waste. The scale constitutes around 5 % of the whole sh (Villamil et al., 2017).
India, with its vast coastline, has a never-ending supply of sh-scale (FS) wastes. The abundant availability and renewability of these scales have made researchers turn their attention toward the valorization of FS waste. Hence, the synthesis of bioplastics was attempted by combining FS wastes and corn starch (CS) using glycerol as a plasticizer. In this study, the major ingredients used were whole discarded FS (irrespective of species) and CS.

Collection of samples
FS were sourced from the shery wastes at Parangipettai sh market in Cuddalore District located in the Southeast coast of India. The scales were collected in zip-lock pouches, which were transported immediately to the laboratory and stored at -20°C until analysis. The other two major ingredients for the study were Corn starch and glycerol (99.5 %). The CS was purchased from the local market and glycerol as well as other analytical chemicals was procured from Merck Ltd., Mumbai, India.

Preparation of sh scales
In the laboratory, the collected FS were cleaned thoroughly with Milli-Q water to remove the impurities.
Furthermore, the FS were treated using different chemical composites, i.e., 1.0 M NaCl, 0.05 M Tris HCl, and 20.0 mM EDTA for 48 h with an adjustable pH of 7.5. This process was intended to remove the proteins and other substances that are attached to the FS (Pati et al., 2010). Subsequently, the cleaned FS were dried in a hot air oven (Technico) at 70 ºC for 24 h, and the dried FS were powdered by using a Fritsch planetary Ball mill (Glen crestan Ltd) until very ne particles were obtained. The obtained ne particles were sieved with a 45µm mesh sieve by using a mechanical sieve shaker (Jayant Gyratory sieve shaker) and again dried in a hot air oven and stored in a desiccator to avoid moisture absorbance for further processing.

2.3.Preparation of lm-forming solution and lm casting
FS and CS powders were the major ingredients for bioplastic lm synthesis. In this study, these two substances were selected at four different ratios, i.e., type1-CS: 10 g pure CS as control, type2-CSFS1: 2.5 g FS and 7.5 g CS, type3-CSFS2: 5 g FS and 5 g CS, type4-FS: 10g FS alone. All the samples were individually prepared by mixing 100 ml of distilled water and 6 ml of glycerol according to the method of Thammahiwes et al. (2017) with a few modi cations. The mixture was heated to 90 ºC and stirred for 30 min using a magnetic stirrer with a hot plate.
The lm-forming solution was cast onto a rimmed silicone resin plate (50×50 mm), and air was blown over the plate for 12 h at room temperature. The resulting lms were manually peeled off and used for further analyses (Arfat et al., 2014
This estimation was done in triplicates, and moisture loss was calculated as follows: Eq (1): Moisture Content (%) = w 1 -w 2 /w 1 ×100 Where, W 1 is the initial weight of the bioplastic lm and W 2 is the nal weight 2.4.3. Opacity UV-VIS Spectrophotometer (SHIMADZU, UV-1800 (Asia Paci c PVT LTD, Singapore) as employed for determining the opacity of the lm. The absorbance of the sample was read at 600 nm (4 cm×1 cm) by measuring light transmittance (Tunc and Duman, 2010). The measurement was performed in triplicates, and opacity was calculated as follows: Opacity (%) = A600 nm /X Where, A600 nm is the absorbance at 600 nm and X is the lm thickness (mm).

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The thickness of the synthesized bioplastic was measured by using a digital vernier caliper. The thickness was checked at different parts of the bioplastic lm, and the average was calculated (Oluwasina et al., 2019).
Average Thickness=Sum of the measured values/number of readings 2.4.5. Solubility in water Solubility determination was carried out according to the method of Lin et al. (2010). Films of 2-cm diameter were incubated in a hot air oven at 105 ºC for 24 h. The weighed samples (wi) were transferred to 50 ml of distilled water and incubated as described. The dry mass (wf) of the samples was determined under prescribed conditions. The solubility (%) of the bioplastic lms was calculated from the results of triplicate experiments.
Eq (3): Where, wi is the initial mass (g) and wf is the nal mass (g).

Fourier transform infrared (FT-IR) spectroscopy
The FT-IR spectra of the bioplastics were analyzed using an FT-IR instrument (Perkin-Elmer Model 1000) tted with an attenuated total re ectance attachment. The spectra were observed at the frequency range 2.4.10.Thermogravimetry/differential thermal analysis (TG-DTA) The samples were subjected to thermal stability testing using a TG analyzer (Netzsch STA449F3) heated gradually at the rate of 10 ºC/min from 100 ºC to 600 ºC under nitrogen atmosphere (Thammahiwes et al., 2018).

Biodegradability test
For biodegradability testing, the assembled bioplastic samples were dried at 45 ºC for 24 h. Samples weighing 5 g were weighed and buried with municipal solid waste fertilizer containing leaves, cow dung, food, paper, wood-waste, composting seeds, urea, and water (Muller, 2005). After 7 days, the bioplastic lms were separated from the municipal compost and SEM images of the specimen were taken after visual examination (Marichelvam et al., 2019). The biodegradability was measured follows: Where, Wo and W are the weights of the samples before and after the test, respectively.

Results
The bioplastic lms prepared with different ratios of FS with CS using the casting method are illustrated in Supplementary Figure 1.
Texture, color, and hot water solubility of the bioplastic lms were tested using sensory evaluation methods, such as visual examination and textural evaluation using the eyes and hands, respectively. The texture of CS was a ne powder, while FS was mildly coarse. CSFS1 and CSFS2 were also mildly coarse powders since FS powder had contributed to their coarseness. The color of CS was pure white, while FS was yellowish white in color. CSFS1 and CSFS2 also appeared as yellowish white powders owing to the presence of FS. When the hot water solubility of CS was tested, it showed gel formation within 15 min of heating, whereas FS was insoluble even after 30 min of heating at 90°C. CSFS1 and CSFS2 formed insoluble gels within 30 min of heating. Similar to texture and color, the CSFS1 and CSFS2 exhibited the property of FS and were insoluble for up to 15 min; however, they later turned into a gel because of the CS content (Table 1).  Figure 1.
The SEM images of CS indicated a rough surface. The adhesion between the CS matrix and the FS powder of the bioplastic lms was also visible in the images. The surface of CSFS1 was smooth and continuous, which is a highly preferred morphology without cavities, edges, or holes. Conversely, the surface of CSFS2 exhibited a rough and irregular surface, which is an unfavorable morphology with cavities. FS surface structure was mildly rough and irregular, which is a slightly unpleasant morphology ( Figure.    XRD studies were performed to analyze the crystalline phase of the bioplastic lms and the patterns. The XRD patterns were examined with 2Ө scans from 10º to 100º. Phase identi cation was achieved by comparing the samples to standards of JCPDS 00-009-0432 for hexagonal hydroxyapatite (HAp) structure (calcium phosphate hydroxide Ca 5 (PO 4   Thermal analysis of the bioplastic lms was conducted using thermogravimetric analysis. TGA of FS possessed the highest thermal stability of 328.8 ºC. However, the thermal stabilities of the other samples were inferior. CSFS1 had a maximum thermal stability of 278.741 ºC, while for CSFS2 and CS it was 273.516 ºC nd 253.7ºC, respectively. The TG curves were associated with a DTA curve to determine the thermal stability. Thermal weight loss was observed in three stages, namely primary, secondary, and tertiary. The primary stage had a temperature range of 0 ºC -300 ºC, while the secondary and tertiary stages had temperature ranges of 300 ºC-350 ºC and 350 ºC-600 ºC, respectively. Sudden maximum weight loss was observed in the secondary stage, while the other stages exhibited gradual and minimal weight loss. Though temperature variations were witnessed in all stages, maximum weight loss occurred in the secondary stage in all the samples (Supplementary Figure 4).
The bioplastic lms were subjected to degradation for a period of 7 days, at the end of which they were visualized using SEM. The surface structure exhibited substantial variations, and the material had lost its evenness. The bioplastic lms presented surface irregularities, which revealed fungal growth and indicated degradation, as witnessed in the SEM images ( Figure. 6). The fungal spores, which were identi ed as Aspergillus sp., caused thinning of the lms and suggested biodegradation. Among all the tested samples, CS exhibited the highest degradation (60 %), followed by CSFS1 (50 %) and CSFS2 (38 %). On the other hand, FS underwent the least degradation (28 %). The percent degradation of all the four sample types is shown in Figure.  The results of sensory evaluation suggested that the CS-based bioplastics were in the form of a ne powder, while the other types (CSFS1, CSFS2, and FS) were granular in nature. Awonusi et al. (2007) stated that the granular nature of FS, CSFS1 and CSFS2 is probably due to the presence of phosphates and amide I and III groups.
The color of the synthesized bioplastics showed that CS type was pure white, while the others were yellowish white. The yellowish white appearance of the FS, CSFS1 and CSFS2 was due to the presence of phosphate groups present in the sh scales (Awonusi et al., 2007).
The hot water solubility test indicated gel formation by CS within 15 min of boiling. CSFS1 and CSFS2 formed insoluble gels, whereas FS was completely insoluble. The reason for the insolubility could not be fathomed, and even previous researchers had encountered di culties in this regard. The water solubility of bioplastic lms is entirely dependent on polymer structure and intermolecular bonds (Kaewprachu and Rawdkuen, 2014). Glycerol is hydrophilic in nature and is used as a plasticizer to ensure bioplastic solubility. In addition, its hydrophilic nature increases the mobility of the peptide chains and contributes to solubility (Ekrami and Emam-Djomeh, 2014). The least solubility of 2.5 % exhibited by FS is lower than that reported by a other studies (Monterrey-Quintero and Sobral, 2000). The dry matter in the lm mesh causes hardness and enhances the traction resistance while reducing its solubility (Neves et al., 2019).
The opacity of the samples is dependent on the composition, origin, and thickness of starch. The greater content of amylose in the samples might be the reason for their higher thickness (Basiak et al., 2017). This observation is in relation to starch. However, Lin et al. (2011) opined that the chief building block of FS is collagen, which also contributes to thickness. In the present study, FS had the highest thickness while CS had the least thickness, which can be correlated to opacity. FS had the highest opacity (8.05 ± 0.08 %), followed by CSFS2 (4.14 ± 0.16 %), CSFS1 (2.45 ± 0.08 %), and CS (2.16 ± 0.06 %). Zavareze et al. (2012) explained the importance of opacity in the coating of food surfaces. However, transparent lms have applications as stressed by Santana et al. (2018). Decreased opacity indicates higher transparency, alluding that CSFS1 is the most suitable owing to its transparency.
Water absorption kinetics of the bioplastic lms reveals the moisture tolerance and the lling of water within the lms. CS displayed the highest absorption (50-70 %), followed by CSFS1 (30-50 %), CSFS2, and FS (20-40 %) within 0-22 min. The higher absorption kinetics leads to the swelling of the lms, which opens more pores and contributes to further accumulation of water. However, the ne powdery nature prevents the excess storage of water. Meanwhile, the mild granular powders facilitate gradual absorption but have high water storage capacity in their pores. These results are in agreement with earlier works on methyl cellulose-montmorillonite lms (Tunc and Duman, 2010) and whey proteins (Galus and Kadzi, 2016).
SEM micrographs showing intact starch granules signi ed incomplete gelatinization during the lm formation. Cross-sections of the lms revealed the presence of an irregular structure with ridges and grooves. These grooves may be the result of microbubble formation during the gelatinization, which was also present in the bioplastic lms synthesized by Santana et al. (2018). Minor discontinuities were observed in all the SEM images at 500 x magni cation, and these discontinuities were identi ed as cracks. These cracks may compromise the structural integrity, thereby causing alterations in functional properties (Souza et al., 2004).
In this study, FS demonstrated higher tensile strength when compared with CSFS1, CSFS2, and CS. Elimination of the unrequired organic compounds and collagen from the FS powder enhanced the mechanical and thermal properties of the lms (Chiarathanakrit et al., 2018). An increase in the amount of FS powder resulted in a slight decrease in the tensile strength of the wheat gluten-based bioplastic lm (Thammahiwes et al., 2017). Bioplastics should have su cient tensile strength to ensure their integrity when used as packaging materials.
FT-IR analysis of HAp powder was performed in the range of 4,000-500 cm −1 to detect the presence of various functional groups. The results were supported by the FT-IR spectra (Figure 7).Wave numbers of 1645, 1546, and 1647 cm −1 were assigned as the peaks for amides I, II, and III of collagen type I The relationship between the temperature and properties of the synthesized bioplastic lms was assessed. The major constituents of FS are collagen, connective tissue proteins, and proteinaceous molecules such as guanine, glycine, alanine, and hydroxyproline (Mondal et al., 2010). TGA curves of the bioplastic lms showed thermal stability in the range of 250°C-330°C. The TG-DTA plots of the samples showed weight loss in three stages, as previously reported (Sobczak-Kupiec and Wzorek, 2012). Weight loss in the primary stage is generally due to the loss of bound water. The weight loss in the secondary stage is due to a complex process that involves saccharide ring dehydration and chitosan depolymerization and denaturation (Wan et al., 2009). Total denaturation of the polymers occurs, which leads to complete weight loss in the tertiary stage (Wan et al., 2009). In the secondary stage, complete decomposition of starch and organic compounds present in the FS powder occurs, which results in maximum weight loss. Similar weight loss pattern was observed in a previous work that focused on bioplastic lms from shrimp shell waste and wheat gluten (Kumari et al., 2016).A number of other works on FS and collagen also reported weight loss during the secondary stage in a temperature range of 250°C-330°C. Their ndings disclose that weight loss in this stage could be due to the cleavage of organic components (Tampieri, et al., 2003).
In earlier studies by Thammahiwes et al. (2017), FS of Lates calcarifer in combination with wheat gluten was used. The maximum observed tensile strength identi ed in their research was 7.51 MPa. In the present investigation, we observed the highest tensile strength of 10.1 ± 0.05 MPa by using corn starch based bioplastics in multiple sh scles.
Biodegradability is a key feature of next-generation plastics, and the European Committee for Standardization speci es 90 % degradation within 6 months (Soroudi and Jakubowicz, 2013). In the biodegradability test, degradation was noticed after 7 days of treatment with organic waste. Degradation was affected by the growth of Aspergillus sp. The SEM images showed thinning of the bioplastic lms mediated by the Aspergillus sp. spores, which con rms the biodegradable nature of the synthesized bioplastic lms. In this case, the fungus facilitating the degradation was naturally present in the organic waste. However, in an earlier study (Abdullah et al., 2018), A. niger were arti cially introduced. These ndings indicate that bioplastics can be naturally degraded without the use of chemicals or other methods of degradation such as photodegradation. Conventional plastics meant for single use can be replaced with these easily degradable bioplastic lms.
In the present study, CS showed the fastest degradation (12 days) while FS showed slowest degradation (no.of days). CSFS1 (no.of days) and CSFS2 (no.of days) showed moderate rates of degradation. The complex polymer chains experienced enzymatic cleavage and were subsequently reduced to short chains of monomers, dimers, and oligomers. These shortened chains act as carbon sources by easily passing through the bacterial membranes. Increased availability of carbon in CS could enhance biodegradation by hydrolysis and subsequent molecular weight loss. The resultant smaller molecules are susceptible to enzymatic attack, causing rapid biodegradation (Bhardwaj et al., 2013).
In a former study by Abdullah et al. (2019), the fungal species A. niger was introduced arti cially to induce biodegradation. However, in this study, Aspergillus sp. naturally present in the soil caused the degradation. This result indicates that bioplastics can be naturally degraded without the addition of chemicals or other inducers.

Conclusion
In this research, FS and CS were used in different combinations to synthesize bioplastic lms. FS and CS applied in the ratio of 1:3 exhibited excellent physical, chemical, and mechanical properties. In addition, the study con rmed that the synthesized plastics are easily biodegradable. Therefore, these bioplastics can be used as viable alternatives to the hazardous petroleum-based conventional plastics. The ecofriendly bioplastics can be used an agricultural eld. However, standardization of technology is required prior to commercial production.

Declarations
Ethics approval and consent to participate: Not applicable