Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=lsrt21

Inorganic and Nano-Metal Chemistry

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lsrt21

Synthesis of biodegradable semolina starch plasticfilms reinforced with biogenically synthesized ZnOnanoparticles

Muhammad Imran Din , Rimsha Sehar , Zaib Hussain , Rida Khalid & AsmaTufail Shah

To cite this article: Muhammad Imran Din , Rimsha Sehar , Zaib Hussain , Rida Khalid &Asma Tufail Shah (2020): Synthesis of biodegradable semolina starch plastic films reinforcedwith biogenically synthesized ZnO nanoparticles, Inorganic and Nano-Metal Chemistry, DOI:10.1080/24701556.2020.1813768

To link to this article: https://doi.org/10.1080/24701556.2020.1813768

Published online: 02 Sep 2020.

Submit your article to this journal

Article views: 9

View related articles

View Crossmark data

Synthesis of biodegradable semolina starch plastic films reinforced withbiogenically synthesized ZnO nanoparticles

Muhammad Imran Dina , Rimsha Sehara, Zaib Hussaina, Rida Khalida, and Asma Tufail Shahb

aInstitute of Chemistry, University of the Punjab, Lahore, Pakistan; bIRCBM, COMSATS University Islamabad, Lahore, Pakistan

ABSTRACTIn present work, Semolina being used as biomass served to be the polymer matrix within whichZnO nanoparticles were incorporated. ZnO nanoparticles were biogenically synthesized usingSyzygium cumini extract to avoid any hazardous side products. FT-IR analysis of ZnO reinforcedSemolina plastic blends revealed hydrogen bond formation between the starch polymer matrixand nanoparticles. Moisture content test was performed which proved the decrease in the mois-ture content with an increase in ZnO NPs concentration where 9.7% served to be the lowest valueby 10% ZnO blend. The water solubility test also confirmed this decreasing trend while increasingwater resistance and antimicrobial activity was also confirmed. The cell cytotoxicity test was per-formed on the fibroblast cells where the 5% ZnO blend confirmed to be cytotoxic. The biodegrad-ation of the 5% ZnO blend confirmed the faster rate of mineralization at the start whiledecreasing through time.

ARTICLE HISTORYReceived 16 May 2020Accepted 26 July 2020

KEYWORDSBiodegradable plastic;starch; semolina;plasticizers; fillers;nanoparticles

Introduction

Recently, natural biopolymers have gained a huge interest inthe progress of eco-friendly packaging materials due to highbiodegradability, sustainability, biocompatibility, and edibility.[1]

Hence lipids, polysaccharides, and protein based materials aregenerally used as biopolymers. Among them, polysaccharidesincluding alginate, chitosan, pectin, carrageenan, starch, agar,and cellulose are particularly attractive due to good film-forming properties and moderate mechanical strength.[2,3]

However, these biopolymer based packaging materials havesome limitations including poor barrier or mechanicalproperties along with high production cost as compared tocommodity plastic films.[4] To overcome these shortcomingsbiopolymers make a hybrid with nanofillers materials that leadto the improvement in mechanical or physical properties offilms. However, the reinforcement of nanofillers into the poly-mer matrix follows ex-situ or in-situ methods.[5] In later, metalnanoparticles are prepared inside the polymer solution whilein former nanofillers are first synthesized and then dispersedinto the polymeric matrices. The ex-situ approach is generallypreferred for the synthesis of efficient polymer bio-nanocomposites.

Among various nanomaterials, zinc oxide nanoparticles(ZnO NPs) have gained huge interest as versatile inorganicfillers because of large surface area, high surface reactivity,high thermal stability, unique physical and mechanical prop-erties.[6–8] Numerous chemical methods have been reportedfor the preparation of the ZnO NPs but these methods havesome major drawbacks like multi-step procedures, costly set-ups, an extreme working environment like high temperature

and pressure, etc. associated with them.[9] Alternatively,green routes are emerging as the new and safest method forthe preparation of the nanoparticles in the past few deca-des.[10] Hence, green synthesis of ZnO NPs from a plantextract of Syzygium cumini leads to the formation of eco-friendly, safe nanoparticles with effective stability.[11–16]

Syzygium cumini (Jamun) is renowned for its great nutri-tional value.[17] Its leaves are famous for their application inayurvedic medicine. The aromatics and phenolic species pre-sent in the leaf extract of S. cumini serve a dual purposeand not only reduce the zinc precursor salt into ZnO NPsbut also stabilize them.

Oleyaei et al.[18] fabricated starch films by integratingTiO2 nanoparticles into the potato starch polymer matrix.Kanmani and Rhim[19] developed bio-nanocomposite filmsthrough solvent casting techniques based on different bio-polymers (agar, carrageenan, or CMC) reinforced with ZnONPs. Sanuja et al.[20] and Jayasuriya et al.[21] prepared chito-san based bio-nanocomposites by integrating ZnO NPs.

In this study, the ZnO reinforced Semolina films havebeen prepared using solution casting method through ex-situ approach. Semolina grains are transparent, hard, tinted,and exhibit antioxidant activities.[22] Semolina chemicallyconstitutes about 70% carbohydrates of which usually60–70% is starch, 25–29% is amylose while 12–14% is pro-tein.[23] Hence, some properties including cost-effectiveness,high carbohydrate content, and high antioxidant activitiesmake semolina a suitable polymer matrix for the productionof bio-nanocomposite. Lin et al.[24] reported that semolinaexhibits exceptional properties for the production of the

CONTACT Muhammad Imran Din imrandin2007@gmail.com Institute of Chemistry, University of Punjab, Lahore-54590, Pakistan� 2020 Taylor & Francis Group, LLC

INORGANIC AND NANO-METAL CHEMISTRYhttps://doi.org/10.1080/24701556.2020.1813768

edible film with ZnO nanomaterials but still it remainspoorly understood. Increased interfacial interaction in formof hydrogen bonds between ZnO nanofiller and polymermatrices like starch, sago starch, PVA, starch/lysine, andsemolina with enhanced mechanical properties also has beenreported.[4,25–29] However, limited research has beenobserved with Semolina as a biopolymer matrix while litera-ture reports incorporation of chemically synthesized ZnOnanorods.[4] To the best of author knowledge, no onereported the reinforcement of biogenically prepared ZnONPs into Semolina polymer matrix for the preparation ofbiodegradable plastic films. This study aims to elect stableand safe nanoparticles as nanofillers in the polymeric matrixwith antimicrobial properties to enhance biodegradable plas-tic films. To study the effects of different concentrations ofbiogenically synthesized ZnO NPs, the films were character-ized by FT-IR analysis. Additionally, their properties likethickness, water solubility, moisture content, cell cytotox-icity, soil bio-degradability were inspected.

Experimental

Materials

Zinc nitrate hexahydrate Zn(NO3)2.6H2O (as Precursor salt),Sodium Hydroxide NaOH (Stabilizer, a base), Deionizedwater (as Primary plasticizer), Glycerin C3H8O3(Secondaryplasticizer), Vinegar CH3COOH (Co-plasticizer), EthanolC2H5OH (Disinfectant). Analytical grade chemicals are beingused in the present research work i.e., they do not need anysupplementary treatment, as they are obtained from Aldrichchemicals. Syzygium cumini (jamun) leaves were collectedin the fresh condition during spring season February-March,2019 from Punjab University Quaid-e-Azam CampusLahore, Pakistan. Leaves were cleaned properly after collec-tion to get rid of dust particles. Semolina was obtained fromthe local market in Lahore, Pakistan. Semolina flour wastaken and further ground. Then it was sieved twice using asieve with mesh no. 80 to obtain a fine powder.

Preparation of ZnO NPs

For the preparation of 10% aqueous leaf extract, 10 g of theleaves were weighed. Weighed leaves were then cut intosmall pieces and were soaked overnight in 100ml de-ionizedwater. Pieces of leaves were grounded using pestle and mor-tar until they took the form of a paste. The grounded leavespaste was then poured into 100ml de-ionized water. Thenthe mixture was boiled and stirred on a hot plate at70–80 �C for15–20min.to guarantee complete extraction.The mixture was then filtered through Whatman’s filterpaper. The extract obtained was then stored in a refrigeratorat 4 �C with proper labeling. About 1M solution of zincnitrate hexahydrate (Zn(NO3)2.6H2O) 50ml of 10% plantextract was prepared and mixed. The mixture was stirredthoroughly, and its pH was checked using a pH meter. ThepH of the mixture was adjusted to 13 by using a2M NaOHsolution, as the pH of the mixture was acidic (� 4.63). Then

the mixture was heated on a hotplate with vigorous stirringat 75–80 �C for 30–40min. The resulting mixture was thenfiltered using Whatman filter paper 1. The residue obtainedwas then oven dried for 2 hr at 60–70 �C. Finally, ZnO NPswere obtained and were stored in airtight sample bottles atroom temperature. The formation of nanoparticles was con-firmed through UV and FT-IR analysis.

Preparation of films

The biodegradable plastic film have been prepared usingsolution casting technique.[30] A titration flask was taken10 g of semolina flour, 6ml of glycerol, 4ml of vinegar, and150ml of de-ionized water were added into it. The wholemixture was stirred thoroughly on a hotplate with the tem-perature slowly increasing up to 80 �C for 45–50min. andthe momentous increase in the viscosity was checked peri-odically. The resulting plastic solution was cooled to theroom temperature and casted into the Teflon plates. TheTeflon plates containing the plastic solution were then ovendried for 16–18 hr at 60 �C, followed by desiccating for3–4 days with a solution of NaBr. The simple semolina filmsthus obtained were stored in airtight plastic bags at roomtemperature. ZnO reinforced Semolina starch nanocompo-site films were also prepared in an above-mentioned way,with the de-ionized water being replaced by the suspensionof nanoparticles with the de-ionized water; the suspensionwas sonicated for about 5–10min. in a sonicator before fur-ther processing (Figure 1). ZnO blends were prepared of dif-ferent concentrations; the amount of ZnO NPs was taken as1%, 3%, 5%, 7%, and 10% of the weight of semolina beingused i.e., 10 g (Table 1). During the preparation of films,ethanol was sprayed and then dried on all apparatuses incontact with the plastic solution especially Teflon plates. Thefilms were taken off from the plates using lab gloves toavoid contamination and fingerprints.

Characterization

UV-VIS analysis and Fourier transform infrared spectros-copy (FT-IR) techniques were used for the analysis of pre-pared ZnO NPs. The reducing functional groups in theextract were confirmed through analysis using these techni-ques. The thickness of the films was measured by using adigital micrometer (Micro-masterVR ) which was accurate toabout 0.001mm according to the method mentioned in lit-erature.[31] The thickness of films of all ZnO NPs composi-tions was observed. Moreover, the characterization of ZnOreinforced Semolina blends also includes water resistancetest, moisture content, and water solubility test. An initialwater resistance test was performed using two of the sam-ples. The test was performed at room temperature i.e., 25 �Cmaintained in dark. The main objective of this test was tocheck the resistance and stability of simple or blended filmtoward the water and local transmission of microbes for21 days by placing them in de-ionized water in Petri disheswhile covering them properly.

2 M. I. DIN ET AL.

Moisture content and water solubility

Moisture content test of the simple, as well as that of theblended films, was carried out by calculating the initialweight of the films and thereby putting them in an oven at110 �C till no significant reduction in the weight of the filmswas detected further. The observations were noted in tripli-cate. The water solubility of two of the films was performedaccording to the method reported by Kotharangannagariet al.[28] The standard 5% ZnO film and simple Semolinafilm was dried by the method explained above i.e., in theoven at about 110 �C till no reduction in the weight wasobserved. Then the initial weight of the dried films wasdetermined and was dipped in a beaker containing about50ml of de-ionized water, keeping the assembly airtight, atroom temperature for about 24 hr while agitating it regu-larly. The whole assembly was kept in dark. The temperatureof the de-ionized water was 23 �C. After performing the teststhrice, solubility was found using the following equation.

Solubility ð%Þ ¼ Initial dry weight – Final dry weight

� 100=Initial dry weight

Cell cytotoxicity experiment

Cell cytotoxicity of the simple Semolina starch film and 5%ZnO blend was evaluated through the cell culture test by thecell adhesion method. A control completely compatible withthe culture cells was used. Viability of the L-929 fibroblastcells was observed for both the films according to the methodreported in the literature.[32,33] The films with 0.5 cm diameter

were placed in 6-well polystyrene cell culture plates. The fibro-blast cells were then maintained and seeded at 2� 104 cells inDMEM. The assembly for both samples was incubated for aculture period of 24 hr at 37 �C and 5% CO2. The films werethen washed with Phosphate buffered saline (PBS), fixed inmethanol, and were observed under a microscope.

Biodegradation test

The biodegradation test was performed according to theamount of CO2 evolution. The soil was collected from thebotanical garden of Punjab University, Lahore Pakistan. Thesoil was enriched with compost and salt solution containing0.2 g KH2PO4, 0.1 g MgSO4, 0.4 g NaNO3, 0.2 g Urea, 0.4 gNH4Cl per kg of soil was added. The pH of the soil wasnoted by determining the pH of the mixture of soil and de-ionized water. A standard film of a 5% ZnO blend was usedfor the test. The film to be analyzed was weighed and wasmixed with 200 g of soil in a sealed 1000ml glass jar. Onejar was also placed without any material inside which servedas a blank while one jar was placed with the referencematerial. The test was carried for blank and as well as forthe reference and was placed in the dark at room tempera-ture i.e.25 ± 2 �C. The water content of the soil was adjustedat about 15.2%, while the content was measured as weightloss. A CO2 trapping solution, about 30ml of 0.5M KOH ina 50ml beaker was positioned in each jar. Then 0.5N HClsolution was used for titration of KOH solution to obtainthe amount of CO2 trapped. The amount of CO2 evolved ingrams was calculated and percentage mineralization wascalculated.[34]

Results and discussion

Structural characterization

The Ultraviolet Visible characterization of ZnO NPs wascarried out to confirm the formation of nanoparticles byestimation of the peak with a maximum wavelength ofabsorption. The UV/VIS spectrometer is employed for theanalysis that can display the absorbance of the sample

Table 1. Representation of blend composition.

ZnO NPs reinforcedSemolina BlendType (%)

Amount of ZnO NPsused per 10gof Semolina (g)

1 0.13 0.35 0.57 0.710 1

Figure 1. Preparative scheme for hybrid nanocomposites.

INORGANIC AND NANO-METAL CHEMISTRY 3

within the range of 200–800 nm. The analysis was performedby dispersing the nanoparticles in deionized water. Figure 2shows that a strong absorption band was observed at355 nm which is related to the intrinsic bandgap of Zn-Oabsorption attributed to the electronic transition from thevalence to the conduction band. This sharp significantabsorption indicates the monodispersed distribution of thenanoparticles.[35] The data reported from the previous

literature thus supports the formation of ZnO NPs. A simi-lar absorption band was found by Talam et al.[36] whichconsequently confirmed the results. Hence surface plasmonresonance at 355 nm suggests the formation of stable ZnONPs without any aggregation.

FT-IR analysis was performed to confirm the functionalgroups of biogenically synthesized ZnO NPs. Generally, finger-print region of the spectrum constitutes the metal oxides peak

Figure 2. Absorption spectra of ZnO nanoparticles.

Figure 3. FT-IR spectrum of ZnO nanoparticles.

4 M. I. DIN ET AL.

which is less than 1000 per centimeters. FTIR spectrum forZnO NPs show peaks at 500–600, 1574, 2888, 2929, 1574 and1335 cm�1 (Figure 3). A sharp peak at 559 cm�1 shows thedeformation of the ZnO bond while the peak at 1547 cm�1

shows its stretching. Symmetric and asymmetric vibrations ofthe C-H bond appear at 28888 and 2929 cm�1 while the C-Obond appears at 1574.39 and 1353.91 cm�1.[37–39] The FT-IRanalysis of the Semolina starch film and consequently the ZnONPs reinforced blends was carried out to confirm the presenceof functional groups and the effect of the subsequent reinforce-ments on the peak values of the spectra. As a result of theinteractions between the polymer matrix and the nanoparticlefillers the peak values of the polymer matrix change.

Figure 4 showed the FT-IR analysis of the Semolinastarch film and different concentrations of ZnO NPs rein-forced blends. The shift in the peaks confirms interactionsbetween the polymer matrix and the nanoparticle fillers. Thepeak values of the polymer matrix change with the additionof ZnO NPs. Additionally, the -OH absorption band shift tolower value from 3280.59 cm�1due to increase in the num-ber of hydrogen bonds between hydroxyl groups of the poly-mer matrix and ZnO NPs. Figure 4 showed that allcompositions of ZnO blends showed a significant decreasein the -OH absorption band, 3263 cm�1was observed in 3%ZnO Semolina blend. Moreover, the 10% ZnO blend showedincrease in absorption band to about 3297 cm�1. The shiftin the C-H stretching absorption of methyl and methylenegroups from 2931.94 cm�1towards lower absorption bandis attributed to the electrostatic attraction between ZnONPs and the hydroxyl groups of the polymer matrix.Additionally, a similar electrostatic attraction is attributed to-C-O bond stretching of -C-O-C- group thereby significantlyshifting the absorption band from 1078 and 1021 cm�1 ofthe simple Semolina starch film. The shift in the C-Hstretching absorption band from 2931.94 cm�1of simpleSemolina film to 2928.24 cm�1 of 10% ZnO blend confirmedthe electrostatic attraction. A regular shift in -C-O absorp-tion band from 1078 and 1021 cm�1 of simple semolina filmup to 1077 and 1015 cm�1 of 10% ZnO blend further con-firmed ZnO-polymer matrix interactions. A significant shiftin the Zn-O bond deformation peak from 602.59 cm�1 of 1%ZnO blend to the major 598 cm�1 of 10% ZnO blend.[18,40,41]

Polymer-nanoparticle interaction is evident from the regularshifts in the absorption peaks with subsequent ZnO incorpor-ation. All the peaks obtained were referred from the previousliterature for the confirmation and were arranged in the tabu-lar form.[19,42–44] From Figure 4 of the FT-IR analysis, it canbe concluded that with the increase in the concentration ofthe ZnO NPs in the blend the interaction between the nano-particles as fillers and the polymer matrix increases owing tothe hydrogen bond formation between them (Table 2).However, no blend showed a new functional group as a resultof successive nanoparticle integration into the Semolina poly-mer matrix which confirms the van der waals interactionbetween ZnO NPs and polymer matrix thus affecting themechanical properties of the blends.[45,46] The more signifi-cant shifts are observed in C-H stretching vibrations, in O-Hvibrations and, Zn-O deformation vibrations.

The thickness of all the blends was measured. The thick-ness of the films ranged from 0.093 to 0.098mm. However,many studies reported the effect of thickness of films ontheir properties[47] while no significant differences wereobserved between the thicknesses of the films to investigatetheir effects as similarly reported in the literature[48] wherelike results were obtained. The water resistance test was

Figure 4. FT-IR spectrum of (a) simple simple semolina film (b) 1% ZnO NPsblend (c) 3% ZnO NPs blend (d) 5% ZnO NPs blend (e) 7% ZnO NPs blend (f)10% ZnO NPs blend.

INORGANIC AND NANO-METAL CHEMISTRY 5

performed on a preliminary basis to initially observe theantimicrobial characteristics and water resistance of theblend. Hence, the observation could be made using twosamples i.e., simple Semolina starch plastic film and 5%ZnO reinforced Semolina blend as it served to be the aver-age of all concentrations. The assembly was allowed the localtransmission of the microbes i.e., no strain of microbes wasspecifically provided thus allowing attack within local

laboratory conditions. As a result, the Simple Semolina plas-tic film was attacked by the locally transmitted microbesand was destroyed as well as dissolved completely within7 days. Additionally, the 5% ZnO blend resisted microbialattack within the observation period of 21 days whereas64.8% of the blend was dissolved in water (Figure 5). Thisindicated resistance toward enzymatic degradation bymicrobes as ZnO is believed to release hydrogen peroxide

Table 2. FT-IR analysis of the simple and hybrid bionanocomposites.

Type of Vibrations

Peak Values (cm-1)For ZnO NPs Blend

0% 1% 3% 5% 7% 10%

Stretching of O-H groups 3280.59 3280 3263 3280 3278 3297Zn-O Bond Deformation – 602.59 602.22 601.72 601.55 598.04Stretching of C-H groups linked to ring of methane H-atoms 2931.94 2926.38 2923.63 2931.33 2928.65 2928.24Symmetric and Asymmetric -COOH Vibrations 1416, 1651 1415, 1651.13 1415, 1645 1415, 1651 1410, 1647 1408, 1654-C-O Bond stretching of C-O-H group 1150.84 1150.47 1151 1151 1150.31 1149-C-O Bond stretching of C-O-C group 1078, 1021 1078, 1019 1077, 1020 1077, 1024 1077, 1015 1077, 1015

Figure 5. Represents the (a) simple semolina film at day 1 of the test (b) Infected semolina film at day 3 (c) 5% ZnO reinforced semolina blend at day 1 (d) 5% ZnOreinforced semolina blend at week 2.

6 M. I. DIN ET AL.

and also generate Zn2þ ions into the cell membranes of themicrobes thus affecting their viability.[41,49] Thus, ZnO NPsintegration into the polymer matrix not only increased waterresistance but also greatly enhanced the antimicrobial prop-erties of blends.

Moisture content and water solubility test

The moisture content inherent within the films not onlymakes the molecular diffusion easier but also enhances thesolubility. The minimization of the moisture content thusproved to be necessary to utilize the films in food packagingapplications.[50] A significant decrease in the moisture contentand water solubility is evident as the number of reinforce-ment increases in the polymer matrix. As the concentrationof filler nanoparticles increases the moisture content decreasesalong with solubility (Figure 6). This reduction is attributed

to the formation of hydrogen bonds between the hydroxylgroup at ZnO NPs surface and polar groups of the polymermatrix thereby enhancing interactions. These interfacial inter-actions decrease the number of available hydroxyl groupswhich makes the films water resistant.[51] Hence, the structureof blend also restricts movement of small molecules in thebiodegradable films of different polymer matrices.[18,45]

Similar results were also reported with ZnO nanorods andnanoparticles in which the increasing hydrophobicity of thefilms was attributed to the hydrophobic character ofZnO.[19,52,53] The initial and final weights of the filmswere observed, and their moisture content was calculated(Table 3). Initial weights were taken and then the films wereoven dried at 110 �C. After complete drying, the final weightswere calculated. Simple Semolina starch film showed max-imum moisture content which was responsible for the hydro-philicity of the film however an increase in ZnO NPsconcentration moisture content significantly decreased withthe least value of 9.7% for 10% ZnO blend. A decrease ofabout 60% was observed in moisture content from simpleSemolina starch film to 10% ZnO blend. Thus, it can be con-cluded that a 10% ZnO blend would represent less hydrophil-icity and improved water resistant properties. The solubilitytest was performed using simple Semolina starch film and the5% ZnO blend as a standard film and then the solubility %was calculated. The percentage solubility of the standard 5%ZnO blend was found to be 34% while simple Semolinastarch film was about 76%. The percentage solubility of thestandard further confirmed the observation that nanoparticlesgreatly enhanced the water resistant properties of theSemolina starch ZnO blends as the blends with a lower per-centage of nanoparticles had greater moisture content wouldexhibit higher hydrophilicity ultimately greater solubility.Thus, increasing the concentration of nanoparticles in theFigure 6. Comparison of moisture content in ZnO blends.

Table 3. Moisture content of all blends.

ZnO Reinforced Semolina Blend Type Initial weight (g) Final weight (g) Moisture Content %

0% ZnO Blend 1.01 0.91 241% ZnO Blend 1.60 1.43 223% ZnO Blend 0.98 0.84 14.25% ZnO Blend 1.47 1.32 10.87% ZnO Blend 1.56 1.40 10.210% ZnO Blend 1.55 1.41 9.7

Figure 7. Representative images of a live/dead assay of cells treated with (a) controlled (b) semolina starch film (c) ZnO starch blend.

INORGANIC AND NANO-METAL CHEMISTRY 7

polymer matrix would decrease the solubility of the blendsin water.

Evaluation of cell cytotoxicity

Figure 7 shows the result of the membrane cytotoxicityexperiment, the simple Semolina starch film and 5% ZnOblend were selected for the comparison based on theresponse which the blend exhibited against the microbialtransmission. It was found that cells were alive in the(Figure 7b) simple semolina starch film, while the film wasbroken implying that the film had no cytotoxic effect on thecells. On the other side the ZnO NPs reinforced film wasintact and all the cells were found dead within the incuba-tion period (Figure 7c). Hence, the ZnO blend was cytotoxicto the fibroblast cells as a result all the cells were dead. Thepresence of ZnO NPs imparted cytotoxic characteristics tothe film and an increase in the nanoparticle concentrationwould consequently increase the cytotoxicity of the blendstoward cells. The cytotoxic character of the 5% ZnOblend is attributed to the generation of Reactive OxygenSpecies (ROS) and to the accumulation of Zn2þ ions in thecytoplasmic region of cells as reported in the literature.[54,55]

However, similar results of the cytotoxicity of ZnO NPswere obtained in previous literature against differentcells.[56,57] Hence it can be concluded that the incorporationof the nanoparticles significantly enhanced the cytotoxicproperty of the films.

Biodegradability test

The biodegradation test was performed using a 5% ZnOreinforced Semolina blend (Table 4). The assembly for

biodegradation was set and analyzed for 15 days for CO2

evolved in grams was calculated and the percentage mineral-ization as shown in Figure 8. The Figure 8 shows well thatthe mineralization trend increased tremendously at first andthen it decreased through time which indicated that the 5%ZnO blend was not resistant toward biodegradation in thesoil in the presence of compost and microorganisms as aresult the stage of mineralization reached quickly whichthen decreased with time. The biodegradation rate was fasterat the initial stages as the CO2evolution in grams wasobserved to be greater where it served to be the metabolicend product.[58–60] Hence it can be concluded that the 5%ZnO semolina blend confirmed to be highly biodegradableand therefore can be used as a packaging material infood packaging.

Conclusion

Biogenically synthesized ZnO NPs from Syzygium cuminileaf extract were reinforced into the Semolina polymermatrix. A sharp absorption peak at 355 nm of UV analysiswhile that of FT-IR at 559 and 1547 cm�1 confirmed theformation of ZnO NPs. According to FT-IR analysis ofblends, a shift in the major peaks of films upon reinforce-ment confirmed interactions between polymer matrix andZnO NPs. 5% ZnO blend resisted the local microbial attackfor 21 days. The moisture content was decreased by 60%from simple Semolina film to 10% ZnO blend the least valuefor which was 9.7%. Water solubility also decreased with anincrease in nanofillers concentration. 5% ZnO blend wasfound to be cytotoxic to fibroblast cells in contrast to simpleSemolina film which did not show any cytotoxicity. Theselected 5% ZnO blend was found to be effectively bio-degradable as the percentage mineralization increased andthen decreased through time. As the prepared Semolina bio-nanocomposites reinforced with biogenically synthesizedZnO NPs were found to be highly biodegradable, waterresistant, and antimicrobial in action, they have the potentialto be used in food packaging applications.

ORCID

Muhammad Imran Din http://orcid.org/0000-0001-7158-2843

References

1. Din, M. I.; Ghaffar, T.; Najeeb, J.; Hussain, Z.; Khalid, R.; Zahid,H. Potential Perspectives of Biodegradable Plastics for FoodPackaging Application-Review of Properties and RecentDevelopments. Food Addit. Contam Part A Chem. Anal. ControlExpo Risk Assess 2020, 37, 665–680. DOI: 10.1080/19440049.2020.1718219.

2. Souza, V. G. L.; Rodrigues, C.; Valente, S.; Pimenta, C.; Pires,J. R. A.; Alves, M. M.; Santos, C. F.; Coelhoso, I. M.; Fernando,A. L. Eco-Friendly Zno/Chitosan Bionanocomposites Films forPackaging of Fresh Poultry Meat. Coatings 2020, 10, 110. DOI:10.3390/coatings10020110.

3. Liu, C.; Huang, J.; Zheng, X.; Liu, S.; Lu, K.; Tang, K.; Liu, J.Heat Sealable Soluble Soybean Polysaccharide/Gelatin Blend

Table 4. Representing the % mineralization of the 5% ZnO blend over15 days.

Day of Analysis CO2 evolved (g) % Mineralization

Day 3 0.64 28.6Day 6 0.55 25.25Day 9 0.45 20.53Day 12 0.38 17.3Day 15 0.26 11.78

Figure 8. Displaying mineralization% Trend of 15 Days.

8 M. I. DIN ET AL.

Edible Films for Food Packaging Applications. Food Packag.Shelf Life 2020, 24, 100485. DOI: 10.1016/j.fpsl.2020.100485.

4. Jafarzadeh, S.; Ariffin, F.; Mahmud, S.; Alias, A. K.; Najafi, A.;Ahmad, M. Characterization of Semolina Biopolymer FilmsEnriched with Zinc Oxide Nano Rods. Ital. J. Food Sci. 2017, 29.

5. Kiro, A.; Bajpai, J.; Bajpai, A. Designing of Silk and Zno BasedAntibacterial and Noncytotoxic Bionanocomposite Films andStudy of Their Mechanical and uv Absorption Behavior. J. Mech.Behav. Biomed. 2017, 65, 281–294. DOI: 10.1016/j.jmbbm.2016.08.029.

6. Ramesan, M.; Jayakrishnan, P.; Sampreeth, T.; Pradyumnan, P.Temperature-Dependent ac Electrical Conductivity, ThermalStability and Different dc Conductivity Modelling of Novel Poly(Vinyl Cinnamate)/Zinc Oxide Nanocomposites. J. Therm. Anal.Calorim. 2017, 129, 135–145. DOI: 10.1007/s10973-017-6140-8.

7. Ramesan, M.; Greeshma, K.; Parvathi, K.; Anilkumar, T.Structural, Electrical, Thermal, and Gas Sensing Properties ofNew Conductive Blend Nanocomposites Based on Polypyrrole/Phenothiazine/Silver-Doped Zinc Oxide. J. Vinyl Addit. Technol.2020, 26, 187–195. DOI: 10.1002/vnl.21732.

8. Ramesan, M.; Siji, C.; Kalaprasad, G.; Bahuleyan, B.; Al-Maghrabi, M. Effect of Silver Doped Zinc Oxide as Nanofillerfor the Development of Biopolymer Nanocomposites fromChitin and Cashew Gum. J. Polym. Environ. 2018, 26,2983–2991. DOI: 10.1007/s10924-018-1187-6.

9. Din, M. I.; Najeeb, J.; Hussain, Z.; Khalid, R.; Ahmad, G.Biogenic Scale up Synthesis of Zno Nano-Flowers with SuperiorNano-Photocatalytic Performance. Inorg. Nano-Met. Chem. 2020,50, 613–619.

10. Din, M. I.; Tariq, M.; Hussain, Z.; Khalid, R. Single Step GreenSynthesis of Nickel and Nickel Oxide Nanoparticles fromHordeum Vulgare for Photocatalytic Degradation of MethyleneBlue Dye. Inorg. Nano-Met. Chem. 2020, 50, 292–297.

11. Hasanuzzaman, M.; Islam, W.; Islam, M. PhytochemicalScreening of Syzygium Cumini (l.) Extracts in Different Solvents.J. Bio-Sci. 2018, 24, 11–14. DOI: 10.3329/jbs.v24i0.37483.

12. Prasad, R.; Swamy, V. S.; Prasad, K. S.; Varma, A. BiogenicSynthesis of Silver Nanoparticles from the Leaf Extract ofSyzygium Cumini (l.) and Its Antibacterial Activity. Int JPharma. Bio. Sci. 2013, 2013, 1–6. DOI: 10.1155/2013/431218.

13. Elansary, H. O.; Salem, M. Z. M.; Ashmawy, N. A.; Yacout,M. M. Chemical Composition, Antibacterial and AntioxidantActivities of Leaves Essential Oils from Syzygium Cumini l.,Cupressus Sempervirens l. And Lantana Camara l. From Egypt.J. Agric. Sci. 2012, 4.

14. Eshwarappa, R. S. B.; Iyer, R. S.; Subbaramaiah, S. R.Antioxidant Activity of Syzygium Cumini Leaf Gall Extracts.BioImpacts 2014, 4, 101–107.

15. Balyan, U.; Verma, S. P.; Sarkar, B. Phenolic Compounds fromSyzygium Cumini (l.) Skeels Leaves: Extraction and MembranePurification. J. Appl. Res. Med. Aroma 2019, 12, 43–58.

16. Azima, A. S.; Noriham, A.; Manshoor, N. Phenolics,Antioxidants and Color Properties of Aqueous Pigmented PlantExtracts: Ardisia Colorata Var. Elliptica, Clitoria Ternatea,Garcinia Mangostana and Syzygium Cumini. J. Funct. Foods2017, 38, 232–241. DOI: 10.1016/j.jff.2017.09.018.

17. Kumar, A.; Ilavarasan, R.; Jayachandran, T.; Deecaraman, M.;Kumar, R. M.; Aravindan, P.; Padmanabhan, N.; Krishan, M.Anti-Inflammatory Activity of Syzygium Cumini Seed. Afr. J.Biotechnol. 2008, 7.

18. Oleyaei, S. A.; Zahedi, Y.; Ghanbarzadeh, B.; Moayedi, A. A.Modification of Physicochemical and Thermal Properties ofStarch Films by Incorporation of tio2 Nanoparticles. Int. J. Biol.Macromol. 2016, 89, 256–264. DOI: 10.1016/j.ijbiomac.2016.04.078.

19. Kanmani, P.; Rhim, J.-W. Properties and Characterization ofBionanocomposite Films Prepared with Various Biopolymersand Zno Nanoparticles. Carbohydr. Polym. 2014, 106, 190–199.DOI: 10.1016/j.carbpol.2014.02.007.

20. Sanuja, S.; Agalya, A.; Umapathy, M. J. Synthesis andCharacterization of Zinc Oxide–Neem Oil–Chitosan

Bionanocomposite for Food Packaging Application. Int. J. Biol.Macromol. 2015, 74, 76–84. DOI: 10.1016/j.ijbiomac.2014.11.036.

21. Jayasuriya, A. C.; Aryaei, A.; Jayatissa, A. H. Zno NanoparticlesInduced Effects on Nanomechanical Behavior and Cell Viabilityof Chitosan Films. Mater Sci Eng C Mater Biol Appl. 2013, 33,3688–3696. DOI: 10.1016/j.msec.2013.04.057.

22. Abdenour, Y.; Mourad, L.; Sihem, T.; Zakia, A.; Ghania, O.Physicochemical and Rheological Properties and Bread-MakingPotential of Durum Flour and Semolina. J. Food Agric. Environ.2017, 15, 14–20.

23. Oladunmoye, O. O.; Aworh, O. C.; Maziya-Dixon, B.;Erukainure, O. L.; Elemo, G. N. Chemical and FunctionalProperties of Cassava Starch, Durum Wheat Semolina Flour, andTheir Blends. Food Sci. Nutr. 2014, 2, 132–138. DOI: 10.1002/fsn3.83.

24. Lin, O. H.; Akil, H. M.; Mahmud, S. Effect of ParticleMorphology on the Properties of Polypropylene/NanometricZinc Oxide (pp/Nanozno) Composites. Adv. Compos. Lett. 2009,18, 096369350901800. DOI: 10.1177/096369350901800302.

25. Akhavan, A.; Khoylou, F.; Ataeivarjovi, E. Preparation andCharacterization of Gamma Irradiated Starch/Pva/ZnoNanocomposite Films. Radiat. Phys. Chem. 2017, 138, 49–53.DOI: 10.1016/j.radphyschem.2017.02.057.

26. Mirjalili, F.; Yassini Ardekani, A. Preparation andCharacterization of Starch Film Accompanied with ZnoNanoparticles. J. Food Process Eng. 2017, 40, e12561. DOI: 10.1111/jfpe.12561.

27. Nafchi, A. M.; Nassiri, R.; Sheibani, S.; Ariffin, F.; Karim, A.Preparation and Characterization of Bionanocomposite FilmsFilled with Nanorod-Rich Zinc Oxide. Carbohydr. Polym. 2013,96, 233–239. DOI: 10.1016/j.carbpol.2013.03.055.

28. Kotharangannagari, V. K.; Krishnan, K. Biodegradable HybridNanocomposites of Starch/Lysine and Zno Nanoparticles withShape Memory Properties. Mater. Design 2016, 109, 590–595.DOI: 10.1016/j.matdes.2016.07.046.

29. Ramesan, M. T.; Varghese, M.; P, J.; Periyat, P. Silver-DopedZinc Oxide as a Nanofiller for Development of Poly (VinylAlcohol)/Poly (Vinyl Pyrrolidone) Blend Nanocomposites. Adv.Polym. Technol. 2018, 37, 137–143. DOI: 10.1002/adv.21650.

30. Ostafi�nska, A.; Mike�sov�a, J.; Krej�c�ıkov�a, S.; Nevoralov�a, M.;�Sturcov�a, A.; Zhigunov, A.; Mich�alkov�a, D.; �Slouf, M.Thermoplastic Starch Composites with tio2 Particles:Preparation, Morphology, Rheology and Mechanical Properties.Int. J. Biol. Macromol. 2017, 101, 273–282. DOI: 10.1016/j.ijbio-mac.2017.03.104.

31. Rojas-Gra€u, M. A.; Raybaudi-Massilia, R. M.; Soliva-Fortuny,R. C.; Avena-Bustillos, R. J.; McHugh, T. H.; Mart�ın-Belloso, O.Apple Puree-Alginate Edible Coating as Carrier of AntimicrobialAgents to Prolong Shelf-Life of Fresh-Cut Apples. PostharvestBiol. Technol. 2007, 45, 254–264. DOI: 10.1016/j.postharvbio.2007.01.017.

32. Savaris, M.; Braga, G. L.; dos Santos, V.; Carvalho, G. A.;Falavigna, A.; Machado, D. C.; Viezzer, C.; Brandalise, R. N.Biocompatibility Assessment of Poly (Lactic Acid) Films afterSterilization with Ethylene Oxide in Histological Study in Vivowith Wistar Rats and Cellular Adhesion of Fibroblasts in Vitro.Int. J. Polym. Sci. 2017, 2017, 1–9. DOI: 10.1155/2017/7158650.

33. Zia, K. M.; Zuber, M.; Barikani, M.; Hussain, R.; Jamil, T.;Anjum, S. Cytotoxicity and Mechanical Behavior ofChitin–Bentonite Clay Based Polyurethane Bio-Nanocomposites.Int. J. Biol. Macromol. 2011, 49, 1131–1136. DOI: 10.1016/j.ijbio-mac.2011.09.010.

34. Chinaglia, S.; Tosin, M.; Degli-Innocenti, F. Biodegradation Rateof Biodegradable Plastics at Molecular Level. Polym. Degrad.Stabil. 2018, 147, 237–244. DOI: 10.1016/j.polymdegradstab.2017.12.011.

35. Panchakarla, L.; Govindaraj, A.; Rao, C. Formation of ZnoNanoparticles by the Reaction of Zinc Metal with AliphaticAlcohols. J. Clust. Sci. 2007, 18, 660–670. DOI: 10.1007/s10876-007-0129-6.

INORGANIC AND NANO-METAL CHEMISTRY 9

36. Talam, S.; Karumuri, S. R.; Gunnam, N. Synthesis,Characterization, and Spectroscopic Properties of ZnoNanoparticles. ISRN Nanotech. 2012, 2012, 1–6. DOI: 10.5402/2012/372505.

37. Kumar, H.; Rani, R. Structural and Optical Characterization ofZno Nanoparticles Synthesized by Microemulsion Route. ILCPA2013, 19, 26–36. DOI: 10.18052/www.scipress.com/ILCPA.19.26.

38. Lanje, A. S.; Sharma, S. J.; Ningthoujam, R. S.; Ahn, J.-S.; Pode,R. B. Low Temperature Dielectric Studies of Zinc Oxide (Zno)Nanoparticles Prepared by Precipitation Method. Adv. PowderTechnol. 2013, 24, 331–335. DOI: 10.1016/j.apt.2012.08.005.

39. G�omez-Mart�ınez, D.; Partal, P.; Mart�ınez, I.; Gallegos, C.Rheological Behaviour and Physical Properties of Controlled-Release Gluten-Based Bioplastics. Bioresour. Technol. 2009, 100,1828–1832. DOI: 10.1016/j.biortech.2008.10.016.

40. D�ıaz-Visurraga, J.; Melendrez, M.; Garcia, A.; Paulraj, M.;Cardenas, G. Semitransparent Chitosan-tio2 NanotubesComposite Film for Food Package Applications. J. Appl. Polym.Sci. 2010, 116, NA–3515. DOI: 10.1002/app.31881.

41. Abdullah, A. H. D.; Putri, O. D.; Fikriyyah, A. K.; Nissa, R. C.;Hidayat, S.; Septiyanto, R. F.; Karina, M.; Satoto, R. Harnessingthe Excellent Mechanical, Barrier and Antimicrobial Propertiesof Zinc Oxide (Zno) to Improve the Performance of Starch-Based Bioplastic. Polym-Plast. Technol. 2020, 59, 1259–1267.DOI: 10.1080/25740881.2020.1738466.

42. Wu, Y.; Geng, F.; Chang, P. R.; Yu, J.; Ma, X. Effect of Agar onthe Microstructure and Performance of Potato Starch Film.Carbohydr. Polym. 2009, 76, 299–304. DOI: 10.1016/j.carbpol.2008.10.031.

43. Yu, J.; Yang, J.; Liu, B.; Ma, X. Preparation and Characterizationof Glycerol Plasticized-Pea Starch/Zno–CarboxymethylcelluloseSodium Nanocomposites. Bioresour. Technol. 2009, 100,2832–2841. DOI: 10.1016/j.biortech.2008.12.045.

44. Anitha, S.; Brabu, B.; Thiruvadigal, D. J.; Gopalakrishnan, C.;Natarajan, T. Optical, Bactericidal and Water RepellentProperties of Electrospun Nano-Composite Membranes ofCellulose Acetate and Zno. Carbohydr. Polym. 2012, 87,1065–1072. DOI: 10.1016/j.carbpol.2011.08.030.

45. Jafarzadeh, S.; Ariffin, F.; Mahmud, S.; Alias, A. K.; Hosseini,S. F.; Ahmad, M. Improving the Physical and ProtectiveFunctions of Semolina Films by Embedding a Blend Nanofillers(Zno-nr and Nano-Kaolin). Food Packag. Shelf Life 2017, 12,66–75. DOI: 10.1016/j.fpsl.2017.03.001.

46. Jafarzadeh, S.; Alias, A.; Ariffin, F.; Mahmud, S. Characterizationof Semolina Protein Film with Incorporated Zinc Oxide NanoRod Intended for Food Packaging. Pol. J. Food Nutr. Sci. 2017,67, 183–190. DOI: 10.1515/pjfns-2016-0025.

47. Biduski, B.; da Silva, F. T.; da Silva, W. M.; El Halal, SLdM.;Pinto, V. Z.; Dias, A. R. G.; da Rosa Zavareze, E. Impact of Acidand Oxidative Modifications, Single or Dual, of Sorghum Starchon Biodegradable Films. Food Chem. 2017, 214, 53–60. DOI: 10.1016/j.foodchem.2016.07.039.

48. Zhang, R.; Wang, X.; Cheng, M. Preparation andCharacterization of Potato Starch Film with Various Size of

Nano-sio2. Polymers 2018, 10, 1172. DOI: 10.3390/polym10101172.

49. Guti�errez, T. J.; Seligra, P. G.; Jaramillo, C. M.; Fam�a, L.;Goyanes, S. Effect of Filler Properties on the AntioxidantResponse of Thermoplastic Starch Composites. Handbook ofComposites from Renewable Materials, Structure and Chemistry;Wiley: Hoboken, NJ, 2016; pp. 337.

50. Bourtoom, T.; Chinnan, M. S. Preparation and Properties ofRice Starch–Chitosan Blend Biodegradable Film. LWT-Food Sci.Technol. 2008, 41, 1633–1641. DOI: 10.1016/j.lwt.2007.10.014.

51. D�ıez-Pascual, A. M.; Diez-Vicente, A. L. Zno-Reinforced Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Bionanocompositeswith Antimicrobial Function for Food Packaging. ACS Appl.Mater. Interfaces. 2014, 6, 9822–9834. DOI: 10.1021/am502261e.

52. Guz, L.; Fam�a, L.; Candal, R.; Goyanes, S. Size Effect of ZnoNanorods on Physicochemical Properties of Plasticized StarchComposites. Carbohydr. Polym. 2017, 157, 1611–1619. DOI: 10.1016/j.carbpol.2016.11.041.

53. Nafchi, A. M.; Alias, A. K.; Mahmud, S.; Robal, M.Antimicrobial, Rheological, and Physicochemical Properties ofSago Starch Films Filled with Nanorod-Rich Zinc Oxide. J. FoodEng. 2012, 113, 511–519. DOI: 10.1016/j.jfoodeng.2012.07.017.

54. Shankar, S.; Teng, X.; Li, G.; Rhim, J.-W. Preparation,Characterization, and Antimicrobial Activity of Gelatin/ZnoNanocomposite Films. Food Hydrocoll. 2015, 45, 264–271. DOI:10.1016/j.foodhyd.2014.12.001.

55. Magesh, G.; Bhoopathi, G.; Nithya, N.; Arun, A. P.; RanjithKumar, E. Effect of Biopolymer Blend Matrix on Structural,Optical and Biological Properties of Chitosan–Agar Blend ZnoNanocomposites. J. Inorg. Organomet. Polym. 2018, 28,1528–1539. DOI: 10.1007/s10904-018-0848-1.

56. Mahendiran, D.; Subash, G.; Selvan, D. A.; Rehana, D.; Kumar,R. S.; Rahiman, A. K. Biosynthesis of Zinc Oxide NanoparticlesUsing Plant Extracts of Aloe Vera and Hibiscus Sabdariffa:Phytochemical, Antibacterial, Antioxidant and anti-ProliferativeStudies. Bionanosci. 2017, 7, 530–545. DOI: 10.1007/s12668-017-0418-y.

57. Balraj, B.; Senthilkumar, N.; Siva, C.; Krithikadevi, R.; Julie, A.;Potheher, I. V.; Arulmozhi, M. Synthesis and Characterization ofZinc Oxide Nanoparticles Using Marine Streptomyces sp. WithIts Investigations on Anticancer and Antibacterial Activity. Res.Chem. Intermed. 2017, 43, 2367–2376. DOI: 10.1007/s11164-016-2766-6.

58. Pang, M. M.; Pun, M. Y.; Ishak, Z. A. M. Degradation Studiesduring Water Absorption, Aerobic Biodegradation, and SoilBurial of Biobased Thermoplastic Starch from AgriculturalWaste/Polypropylene Blends. J. Appl. Polym. Sci. 2013, 129,3656–3664. DOI: 10.1002/app.39123.

59. Shah, A. A.; Hasan, F.; Hameed, A.; Ahmed, S. BiologicalDegradation of Plastics: A Comprehensive Review. Biotechnol.Adv. 2008, 26, 246–265. DOI: 10.1016/j.biotechadv.2007.12.005.

60. Iovino, R.; Zullo, R.; Rao, M.; Cassar, L.; Gianfreda, L.Biodegradation of Poly (Lactic Acid)/Starch/Coir Biocompositesunder Controlled Composting Conditions. Polym. Degrad. Stab.2008, 93, 147–157. DOI: 10.1016/j.polymdegradstab.2007.10.011.

10 M. I. DIN ET AL.