Ecofriendly and Biodegradable Soybean Protein Isolate FilmsIncorporated with ZnO Nanoparticles for Food PackagingSiying Tang,† Zhe Wang,*,‡,§ Wan Li,† Miao Li,‡ Qiuhong Deng,‡ Yi Wang,§ Chengyong Li,∥

and Paul K. Chu*,†

†Department of Physics and Department of Materials Science and Engineering, City University of Hong Kong, Tat Chee Avenue,Kowloon, Hong Kong, China‡Food Science and Processing Research Center, Shenzhen University, Shenzhen 518060, China§Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China∥Shenzhen Institute of Guangdong Ocean University, Shenzhen 518108, China

*S Supporting Information

ABSTRACT: ZnO nanoparticles (ZnONPs) are synthesized and incorporated into soybean protein isolate (SPI) to obtainSPI/ZnONPs (SZ) films, and the morphology, size distribution, and stability are determined. The effects of different contentsof ZnONPs in the SZ films on the oxygen barrier, antibacterial activity, and thermal and mechanical properties are evaluated. AZnONPs content of 0.2% in the SZ films improves the tensile strength and microbial inhibition by 231% and 16%, respectively.The thermal stability and oxygen barrier properties of the SZ films are also enhanced with addition of ZnONPs. The ZnONPsdispersed uniformly in the SPI film enhance the interactions between SPI molecules via hydrogen bonding, and the resultssuggest potential application of ZnONPs in food packaging.

KEYWORDS: soybean protein isolate, ZnONPs, food packaging, mechanical properties, thermal stability

1. INTRODUCTION

Recently, research of food packaging materials has focusedmore on renewable and biodegradable films composed ofproteins, polysaccharides, lipids, or their combinations. Thesefilms are required to have not only good biodegradability oredibility, but also physical protection under practicalconditions as well as a satisfactory shelf life.1,2 Among thevarious types of natural materials, soybean protein isolate(SPI) has attracted much interest due to the low cost, filmformability, emulsification properties, biocompatibility, andbiodegradability. Nevertheless, it tends to have poormechanical properties, water resistance, and heat tolerationthereby stifling wider application in food packaging.3,4

Attempts have been made to improve the toughness of SPIfilm by cross-linking using heat, chemicals, enzymes,irradiation, and nanoparticles.5,6 In particular, addition ofinorganic nanoparticles with a large specific surface area offersadvantages such as the low cost, straightforward procedures,

good antimicrobial activity, and chemical inertness. Theincorporated nanofillers can enhance crystallization of theSPI matrix in addition to the thermal resistance, mechanicalcharacteristics, and barrier properties of the films.7−9

Zinc oxide nanoparticles (ZnONPs) have low toxicity andare chemically inert as well as relatively inexpensive as coatingagents. They also possess attractive catalytic properties,antimicrobial properties, and the capability to enhance thethermal, barrier, and mechanical properties of polymers.10−14

Hence, it has been used in cereal-based food fortification andlisted as GRAS (Generally Recognized As Safe) materials(21CFR182.8991) by the United States Food and DrugAdministration (USFDA) in 2014.14,15 However, having alarge specific surface area and volume effects, bare ZnONPs

Received: February 28, 2019Accepted: April 8, 2019Published: April 8, 2019

Article

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© 2019 American Chemical Society 2202 DOI: 10.1021/acsabm.9b00170ACS Appl. Bio Mater. 2019, 2, 2202−2207

can easy aggregate in the solution and lose the effectiveness.Therefore, immobilization of ZnONPs in films is crucial tomaintaining the desirable properties. In this work, ZnONPs aresynthesized and added to SPI to produce SZ films suitable forfood packaging. The size distribution, morphology, andstability of the ZnONPs are determined and the mechanical,barrier, antimicrobial, and thermal properties of the SZ filmsare evaluated systematically.

2. EXPERIMENTAL SECTION2.1. Materials. The soybean protein isolate (SPI, protein ≥90%)

was acquired from Beijing Solarbio Science and Technology Co., Ltd.and zinc acetate dihydrate (AR), diethylene glycol, sodium hydroxide(AR), and Tween 80 were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. E. coli and S. enteric were provided byGuangdong Institute of Microbiology (Guangzhou, China). All thechemicals were analytical grade, and distilled water was usedthroughout sample preparation.2.2. Synthesis of ZnO Nanoparticles (ZnONPs). The ZnONPs

were produced by hydrolysis of zinc salts in polyol under alkalineconditions.16 In brief, 0.3 g of zinc acetate dihydrate was mixed with15 mL of diethylene glycol and 400 μL of NaOH (0.01M) understirring. The mixture was transferred to an autoclave and heated to180 °C for 2 h. The milky white precipitate (ZnONPs) was collected,cleaned with methanol three times, and dried at 60 °C.2.3. SPI/ZnONPs (SZ) Films Preparation. The films were

fabricated using a reported method with some modifications.1 TheZnONPs solutions with different concentrations of 0.01%, 0.05%,0.1%, and 0.2% (w/v) were sonicated for 25 min and heated to 80 °C.The SPI (4%, w/v) powder was dispersed in the heated ZnONPssolution and stirred for 40 min followed by addition of glycerol (1.0g) and Tween 80 (0.5 g). The solutions were labeled SZ1, SZ2, SZ3,and SZ4, and the solution without ZnONPs was the control. Thesolution (100 mL) was degassed in vacuum for 20 min before thecasting process was conducted on square plastic dishes (20 × 20 cm2).To produce films with a uniform thickness, the dishes were put on alevel surface and dried in a chamber at 60 °C for 6 h. Finally, the filmswere preconditioned in a constant temperature and humidity chamber(25 °C, 50% relative humidity) for at least 48 h to normalize themoisture content before further experiments.2.4. Characterization of ZnONPs. Transmission electron

microscopy (TEM) was performed to examine the ZnONPs on theFEI Tecnai G2 F20 S-Twin instrument (FEI Company, Eindhoven,Netherlands) at 200 kV. The zeta potentials were determined on theZetasizer Nano-ZS90 (Malvern Instruments, UK) to evaluate theparticle stability in the suspension against agglomeration. A foldedcapillary cell (DTS 1070, Malvern Instruments, UK) containing 0.5mL of the ZnONPs suspension (0.5 mL) was measured at roomtemperature and analyzed in triplicate.2.5. Characterization of SPI/ZnONPs (SZ) Films. The surface

and interior morphologies of the films were characterized by scanningelectron microscopy (SEM) and energy-dispersive X-ray spectroscopy(EDS). The samples were cryo-fractured in liquid nitrogen to exposethe cross-section of the film. The samples were mounted on a metalstub and coated with platinum for 15 s (Bio-Rad type SC 502, JEOLLtd., Japan) prior to conducting SEM at 5 kV (FEI NovaNanoSEM450, FEI company, Eindhoven, Netherlands).The infrared (IR) spectra were recorded on an attenuated total

reflectance Fourier-transform infrared (ATR-FTIR) spectrometer(Nicolet Avatar 360, Thermo Nicolet Corporation, USA) from 400to 4000 cm−1 employing an attenuated total reflectance (ATR)accessory with a diamond ATR crystal. X-ray diffraction (XRD) wasperformed on the Bruker Advance D8 (Bruker, Germany) using CuKα radiation (λ = 1.5438 Å) with a step size of 2θ = 0.02°.The film thickness was determined on a micrometer (Mitutoyo

Manufacturing, Tokyo, Japan) with a precision of 0.001 mm. Fiverandom locations were measured on each sample, and the averagethickness was used to calculate the tensile strength. The testometricmachine (PARAM XLW-B Auto Tensile Tester, Labthink, Jinan,

China) was employed to measure the tensile strength (TS, MPa) andelongation at break (E, %) of the films. The test was conducted at 25± 1 °C following the ASTM standard method of D882−01. Briefly,120 mm × 15 mm strips cut from the films were equilibrated in ahumidity chamber at a constant temperature (25 °C, 50% relativehumidity) before the analysis. The strips were fastened with an initialgrip separation of 50 mm before stretching. In the process, the crossspeed was 50 mm/min and data about the strength and elongationwere collected by a microcomputer. At least five measurements wereperformed on each sample. To determine the oxygen permeability(OP), the films were cut into round specimens with a diameter of 97mm before conditioning in the cell of a gas permeability tester G2/132 (Labthink, Jinan, China) for 0.5 h. The OP values of films weredetermined at 23 °C and 50% relative humidity.

The antibacterial activity of the films was examined using E. coli andS. enterica by an inhibition zone method. Sequential 10-fold dilutionwith normal saline was first conducted to form the bacterialsuspensions with concentrations between 105 and 106 CFU/mL. Inthe qualitative experiments for the antimicrobial activity, square films(1.0 × 1.0 cm) were obtained from punching and put onto a modifiedagar diffusion assay. Two-hundred microliters of 106 CFU/mL of theE. coli and S. enterica suspension was dispensed, respectively, onto twoplastic plates containing count agar (PCA, Beijing Land BridgeTechnology Co., Ltd.) and the films were placed directly on the agarplates. The clear zones on the plates after incubation (37 °C, 1 day)were recorded, and the antimicrobial test was performed in triplicate.

The thermal stability was assessed on a thermogravimetric analyzer(TGA/DSC1, Mettler Toledo, Switzerland) by thermal gravityanalysis (TGA) and differential scanning calorimetry (DSC).Approximately 5−10 mg of each sample was put on a standardaluminum pan and heated to 600 °C under flowing nitrogen (50 cm3/min) at a rate of 10 °C/min. The central finite difference method wasutilized to calculate the derivative of TGA and the char content of thesamples at 600 °C was determined from the TGA curve. The DSCmeasurement was carried out at a heating rate of 10 °C/min innitrogen between 20 and 300 °C.

2.6. Statistical Analysis. The data were calculated as the mean oftriplicate measurements. SPSS 10.0 for Windows (SPSS Inc., Chicago,IL, USA) was used to conduct the analysis of variance (ANOVA) andDuncan’s multiple range tests were adopted to determine thesignificant difference among the means. Statistical significance wasdefined as p < 0.05.

3. RESULTS AND DISCUSSION3.1. Morphology and Size Distribution of ZnONPs.

The representative TEM micrograph in Figure 1a shows thatthe ZnONPs are porous spheres and distributed uniformlyafter the ultrasonic treatment. Figure 1c shows that the meandiameter of the ZnONPs particles determined by TEM is 91 ±11 nm. The average zeta potential of the ZnONPs is −31 ± 1.5mV, indicating strong electrostatic repulsion among particles(Figure 1b). The stability of the nanoparticle dispersionsdepends on the interactions between the particles and solventand the negative charges on the ZnONPs surface arise fromzinc salt hydrolysis under alkaline conditions.17 The larger thezeta potential (absolute value), the stronger is the repulsionand more stable the system.18,19 Hence, the absolute zetapotential (above 30 mV) indicates high stability in the aqueousmedium.

3.2. Surface and Internal Morphology of the Film.Figure 2a shows that the SPI film has a compact, smooth, andcontinuous surface demonstrating the good film-formingability. However, SZ3 has a relatively rough surfacemorphology, and Figure 2b shows white aggregates on thesurface. Nevertheless, the two sides of the SZ3 film (Figure2b,c) show completely different morphologies. Figure 2bshows a relatively smooth surface with some substances in the

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SPI matrix. Furthermore, the upper layer in SZ3 consists of SPIand ZnONPs rather than pure SPI according to Figure 3.Figure 2c and d reveal surface globules due to phasesegregation in the SZ films. Many ZnONPs are dispersedand aggregate on the globular surface (Figure 2e), andconnections are observed between two globules (Figure 2f).This observation demonstrates uniform dispersion of ZnONPs

in the SPI matrix and the agglomerates become more regular.Therefore, they are more prone to molecular interactions suchas hydrogen bonding and electrostatic forces in the SPI matrix,resulting in improved mechanical and oxygen barrier proper-ties.

3.3. Molecular Interactions. Figure 4a shows the resultsof ATR-FTIR to investigate the interactions between ZnONPs

Figure 1. Morphology and size distribution: (a) TEM image ofZnONPs; (b) zeta potentials of ZnONPs; (c) size distribution ofZnONPs; (d) fabrication process of the SZ film (scale bars = 10 cm).

Figure 2. SEM micrographs: (a) surface morphology of the SPI film;(b) upper layer in the SZ3 film; (c) bottom layer in the SZ3 film; (d)cross-section of the SZ3 film: (e) globular structure (50kmagnification); (f) globular structure (30k magnification).

Figure 3. EDS spectra of the upper layer in the SZ3 film.

Figure 4. (a) ATR-FTIR and (b) XRD spectra of the SPI-based films.

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and SPI matrix. All of the films show the major peak of amide-Iat 1628 cm−1 as a result of coupled C=O stretching/hydrogenbonding with the COO group. The characteristic band of theplasticizer −OH group interplayed by SPI is around 1042cm−1, which indicates the interaction between glycerol andSPI. With regard to amide-A in the films, a broad peak near3278 cm−1 representing N−H stretching coupled withhydrogen bonding is observed. As for amide-B, the peak near2980 cm−1 arises from asymmetric stretching of CH and NH3

+.The intensity of amide-A (the hydrogen bonded N−Hstretching vibration) around 3278 cm−1 increases afteraddition of ZnONPs. The intensity increase in the amide-Aregion demonstrates that hydrogen bonding is responsible forthe interactions between the N−H groups of the SPI chain andZnO NPs.3.4. Crystalline Structure. XRD is carried out to

determine the crystal structure as well as affinity betweenSPI and ZnONPs. The structure of the SZ films is presented inFigure 4b, and the patterns of the SZ films are similar. Thewide diffraction peak at 2θ = 20° belongs to the secondaryconformation β-sheet of SPI,20 and those at 2θ = 32°, 36°,and 56° correspond to the wurtzite structure of ZnONPs(JCPDS file no. 36−1451) showing good compatibilitybetween ZnONPs and SPI.14

3.5. Mechanical Properties. As shown in Table 1, thethickness of SZ film is quite different (p < 0.05) from that ofthe SPI film, and the thickness is altered by the composition ofthe ZnONPs. The tensile strength (TS) and elongation atbreak (E) of the SPI films are 1.42 ± 0.07 MPa and 136.05 ±19.79%, respectively, and in good agreement with previouslyreported values for SPI films.8,21 The TS of the SZ3 film istwice that of SPI films and increases with increasing ZnONPsuntil 4.7 MPa, which is twice that of the original films. Thestrong interfacial interaction between the ZnONPs and SPImatrix may stem from this phenomenon. However, E decreasesfrom 136.05% to 102% due to the increase in the crystallinityafter addition of 0.2% ZnONPs to the SPI films. Typically,ZnONPs act as effective fillers in the matrix and establishstrong interfacial interactions with the matrix because of thelarge surface area. It has been reported that the interfacialinteraction between ZnONPs and starch, gelatin, and chitosancan be enhanced.15,22

3.6. Oxygen Permeability. With regard to edible polymerfilms in food packaging, the gas permeability is an importantproperty23,24 and influenced by various factors such as the pathtortuosity in the polymer structure, polymer chain immobiliza-tion, crystallinity degree, dispersion, variety, content, add-inratio, and direction of the filler as well as solvent retention andporosity.25−27 The oxygen permeability (OP) values of the SZfilms with various contents of ZnONPs are presented in Table1. The SPI film shows an OP of 3.08 ± 1.47 × 108 cm3 cm/cm2 s Pa, and the SZ film has OP in the range between 1.78 ±

1.09 and 2.90 ± 0.34 × 10−8 cm3 cm/cm2 s Pa. The resultsindicate that addition of ZnONPs to the SPI film slightlypromotes the oxygen barrier property probably due to theincreasing tortuosity that obstructs diffusion of oxygen throughthe film. The water resistance is also a vital indicator in foodpackaging and the permeability tends to decrease withincreasing ZnONPs amounts, but the difference is notsignificant (Table S1).

3.7. Antimicrobial Properties. E. coli and S. enteric, tworepresentative food pollution microorganisms, are chosen toassess the antimicrobial activity of the SZ films in foodpackaging. The results are indicated by the size of theinhibition zone as shown in Figure 5. SPI, SZ1, and SZ2 do not

Table 1. Mechanical Properties and Oxygen Permeability of SPI/ZnONPs Films1

film thickness (μm) TS (Mpa) E (%) OP (10−8 cm3 cm/cm2 s Pa)

SPI 62.2 ± 1.79b 1.42 ± 0.07d 136.05 ± 19.79c 3.08 ± 1.47a

SZ1 79.91 ± 13.02a 2.34 ± 0.12c 123.6 ± 7.75c 1.78 ± 1.09a

SZ2 63.36 ± 8.27a 2.99 ± 0.25b 162.54 ± 2.29b 2.54 ± 0.43a

SZ3 63.14 ± 5.26a 2.74 ± 0.17b 199.63 ± 7.95a 2.90 ± 0.34a

SZ4 58.48 ± 4.68a 4.7 ± 0.52a 102 ± 9.89c,d 1.90 ± 0.78a

1Superscripted a, b, c, d in each measurement, the data marked by different letters in a column indicate a significant difference (p < 0.05) and thevalues are shown as mean ± SD.

Figure 5. Antibacterial activity of the samples against (a) E. coli and(b) S. enterica.

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show any antibacterial activity but on the contrary, the SZ3and SZ4 films exhibit distinctive antimicrobial activity againstboth E. coli and S. enterica. Addition of ZnONPs enhances theantimicrobial activity of the SZ films perhaps due to theirregular and sharp morphology of the ZnONPs as well asoxygen vacancies on the polar facets of the ZnNPs. The sharpedges on ZnONPs help penetration into the bacteria cell walls,while oxygen vacancies enable production of reactive oxygenspecies (ROS) to kill bacteria.28

Besides the concentration dependence, the activity of thesamples for the two microorganisms is quite different. Theantimicrobial activity of the SZ films against E. coli (Gram-negative bacteria) is superior to that against S. aureus (Gram-positive bacteria) as indicated by that both SZ3 and SZ4 showlarger microbial inhibition zones against E. coli than S.enterica.14,29 This difference may be attributed to the cellwall structure. Gram-positive bacteria have a cell wallconstructed with a thick but single peptidoglycan layer,which permits easier entry of ZnONPs into the cell.30,31

3.8. Thermal Stability. Figure 6a and b present the TGAand DSC data. As shown in Figure 6a, the first stage fromroom temperature to 150 °C demonstrates loss of water anddesorption of small molecules from the films. The second stagefrom 150 to 310 °C is the result of glycerol evaporation andthermal decomposition of proteins is observed from the thirdstage from 310 to 500 °C.32 In the DSC curves (Figure 6b),the recrystallization peak at around 110 °C is observed and ataround 170 °C, melting occurs. The films with ZnONPs showbetter thermal stability than the SPI film because of the higherthermal stability of ZnONPs, as evidenced by less decom-position of the SZ films (Figure 6a). Stronger interfacialadhesion between the ZnONPs and SPI matrix leads torestricted motion of the SPI chains giving rise to higherthermal stability of the SZ films.33

4. CONCLUSION

SPI-based films containing different concentrations ofZnONPs are prepared and evaluated. Addition of ZnONPsimproves the properties of the SPI films, especially the tensilestrength (TS) and antimicrobial activity. TS increases by231%, whereas E decreases by 25.03% compared to the pureSPI films. The SZ films have better oxygen barrier propertiesshowing an increase of 42.3% compared to the control.Furthermore, addition of ZnONPs enhances the antimicrobialability and the higher thermal stability of ZnONPs improves

the thermal stability of the SZ films. Our results show that SZfilms have high potential in food packaging.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsabm.9b00170.

Details of WVP values for the films (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: paul.chu@cityu.edu.hk. Phone: [852]-34427724.*E-mail: wzfood@szu.edu.cn. Phone: [86]-13048853377.ORCIDSiying Tang: 0000-0003-3621-6092Yi Wang: 0000-0003-1567-3358Chengyong Li: 0000-0003-0018-165XNotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe would like to thank Shenzhen overseas special fund forhigh-level talents [No. KQJSCX2017033116171850] andHong Kong Research Grants Council (RGC) GeneralResearch Funds (GRF) No. CityU 11205617 for the financialsupport.

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Figure 6. (a) TGA and (b) DSC curves of the SPI-based films, and inset in panel b shows the temperature (Tp).

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S-1

Supporting Information

Ecofriendly and Biodegradable Soybean Protein Isolate Films

Incorporated with ZnO Nanoparticles for Food Packaging

Siying Tang a, Zhe Wang b,c,*, Wan Li a, Miao Li b, Qiuhong Deng b, Yi Wang c,

Chengyong Li d, Paul K. Chu a,*

a Department of Physics and Department of Materials Science and Engineering, City

University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

b Food Science and Processing Research Center, Shenzhen University, Shenzhen

518060

c Department of Applied Biology and Chemical Technology, The Hong Kong

Polytechnic University, Kowloon, Hong Kong, China

d Shenzhen Institute of Guangdong Ocean University, Shenzhen 518108, China

*Corresponding Authors: Paul K. Chu (paul.chu@cityu.edu.hk); Zhe Wang (wzfood@szu.edu.cn)

S-2

Table S1. Water Vapor Permeability (WVP) values for the films.

Sample SPI SZ1 SZ2 SZ3 SZ4

WVP (×10-11gcm-1

s-1 Pa-1)

6.65±0.57a 6.39±0.35a 6.48±0.33a 6.26±0.44a 6.11±0.25a

In each measurement, the data marked by different letters in the column indicate

significant difference (p < 0.05).