Accepted Manuscript Title: Preparation of poly(lactide)/lignin/silver nanoparticles composite films with UV light barrier and antibacterial properties Authors: Shiv Shankar, Jong-Whan Rhim, Keehoon Won PII: DOI: Reference:

S0141-8130(17)32959-8 BIOMAC 8333

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

8-8-2017 22-9-2017 7-10-2017

Please cite this article as: Shiv Shankar, Jong-Whan Rhim, Keehoon Won, Preparation of poly(lactide)/lignin/silver nanoparticles composite films with UV light barrier and antibacterial properties, International Journal of Biological Macromolecules This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Preparation of poly(lactide)/lignin/silver nanoparticles composite films with UV light barrier and antibacterial properties

Running title: PLA/lignin/AgNPs composite films

Shiv Shankar1, Jong-Whan Rhim1,*, and Keehoon Won2


Center for Humanities and Sciences, and Department of Food and Nutrition, Kyung Hee

University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea 2

Department of Chemical and Biochemical Engineering, Dongguk University–Seoul, 30

Pildong-ro 1-gil, Jung-gu, Seoul 04620, Republic of Korea


Corresponding author

Tel: +82-61-450-2423 Fax: +82-61-454-1521 E-mail: [email protected]

Highlights 

Organosolv lignin was used as a reducing agent for the preparation of silver nanoparticles.

AgNPs were reinforced into PLA polymer to prepare composite films.

Mechanical and water vapor barrier properties of the composite films increased.

Composite films exhibited potent antibacterial activity.


Abstract Organosolv lignin was used as a reducing agent for the preparation of silver nanoparticles (AgNPs) and their incorporation into poly(lactide) (PLA) polymer to prepare composite films. The composite films were characterized using UV-visible spectroscopy, FE-SEM, FTIR, XRD, and TGA. The optical, mechanical, water vapor barrier, and antibacterial properties of the composite films were evaluated. The UV-visible spectra of films exhibited two characteristics peaks around 300 and 450 nm attributed to lignin and AgNPs, respectively. XRD results indicated that the crystalline AgNPs had been formed. The transmission of light at 280 nm decreased significantly after incorporation of lignin and AgNPs. FTIR results showed that there was no change in the chemical structure of PLA after incorporation of lignin and AgNPs. The mechanical and water vapor barrier properties of the composite films increased after lignin and AgNPs incorporation, The films containing AgNPs exhibited potent antibacterial activity against Escherichia coli and Listeria monocytogenes.

Keywords: Organosolv lignin; Composite film; PLA; Antibacterial activity 1.

Introduction Conventionally, various types of chemical preservatives have been used to reduce spoilage of

food caused by microorganisms to extend the shelf life of foods. Spraying or adding the antibacterial agents to the food surface or dipping the food materials in an antibacterial solution is the most commonly used methods to inhibit the bacterial growth on the surface of the food [1]. However, such practice of direct inclusion of antimicrobials raised a question on the diffusion of the high amount of antibacterial agents into the food. As an alternative, active packaging


materials with antibacterial activity have attracted great attention for protecting packaged food [2,3]. For this purpose, various types of chemicals, plant extracts, enzymes, bacteriocins, and metallic or metallic oxide nanoparticles have been used. Among them, metallic nanoparticles are promising due to their unique functional properties such as electric, optical, catalytic, thermal stability, and antimicrobial properties [4,5]. Among the metallic nanoparticles, silver nanoparticles (AgNPs) have been most widely used for the preparation of nanocomposite in the food packaging due to their high thermal stability and broad-spectrum of antimicrobial properties [4,6]. Various natural biopolymers have been used to develop antimicrobial food packaging films by blending with AgNPs [7-9]. However, hydrophilic nature of such natural biopolymer-based films has restricted their use in packaging of food with high moisture content. To solve the problem of natural biopolymer-based films, a variety of synthetic biopolymers such as poly(lactide) (PLA), poly(butylene succinate) (PBS), poly(butylene adipate-co-terephthalate) (PBAT), and poly(hydroxy alkanoates) (PHAs) have been suggested to prepare antimicrobial bio-nanocomposite films due to their eco-friendliness, good processability, and acceptable mechanical and barrier properties [10]. Among such synthetic biodegradable plastics, PLA is considered as a promising material due to its full biodegradability, thermoplasticity, high transparency, moderate water resistance, and commercial availability [11]. Since PLA is soluble in chloroform, PLA films have been prepared using a solvent casting method [12]. However, the inclusion of AgNPs into PLA matrix using the solvent casting method is difficult since AgNPs synthesized in an aqueous solution is not compatible with the organic solvent for the dissolution of PLA [8,13]. Shankar & Rhim proposed a synthesis of AgNPs in chloroform using tocopherol as a reducing and capping agent for the preparation of PLA/AgNPs composite film [8]. However,


use of tocopherol for the food packaging application is not practical due to its high cost and availability. Lignin is the second most abundant renewable and biodegradable natural polymer next to cellulose in the plant kingdom. It contains many functional groups in different proportions in the structure, having a high potential for the chemical modification and adjustment of polarity to increase the compatibility with appropriate polymeric matrices [14-16].

It has been

demonstrated that the use of lignin as a filler enhanced the thermal and mechanical properties of polymer matrices [17,18]. Also, lignin possesses antioxidant and UV screening property [19-21]. Lignin can be obtained from pulping processes as well as from its botanical source, which results in technical lignins. There are four different types of technical lignins: lignosulfonate, kraft, soda, and organosolv lignin. Unlike the other technical lignins, organosolv lignin, which is extracted through pulping with organic solvents, is soluble in a wide range of organic solvents [20]. Also, lignin possesses multiple functional groups such as reductive aliphatic hydroxyls, phenolic hydroxyls, and thiols, which can serve as reducing and stabilizing agents for the synthesis of silver nanoparticles [2]. Therefore, organosolv lignin could be a good candidate as a reducing and capping agent for the preparation of PLA/AgNPs composite films in organic solvent (chloroform) as a cosolvent for the organosolv lignin and the polymer matrix (PLA). To the best of our knowledge, no reports are available on the synthesis of silver nanoparticles using chloroform as a cosolvent for PLA and lignin as reducing and capping agents.

The main objective of the present study is to prepare solvent cast PLA/AgNPs composite films using organosolv lignin in an organic solvent for dissolving PLA with homogeneous blending. The composite films were characterized using various analytical techniques, and the


UV-light barrier, mechanical, thermal, water vapor barrier, and antibacterial properties of the composite films were also evaluated.


Materials and methods


Materials PLA (PLLA, Biomer® L9000; average molecular weight = 200 kDa) was procured from

Biomer Inc. (Krailling, Germany). PLA resins were dried under vacuum at 60 °C for 24 h before use. Organosolv lignin (Mw: 4130 Da, Mn: 1200 Da, PDI: 3.44) was purchased from SigmaAldrich (St. Louis, MO, USA). Silver nitrate (AgNO3), brain heart infusion broth (BHI), and tryptic soy broth (TSB) were obtained from Duksan Pure Chemicals Co., Ltd. (Gyeonggi-do, Korea). Chloroform was procured from Daejung Chemicals & Metals Co., Ltd. (Siheung, Gyeonggi-do, Korea). Escherichia coli O157: H7 ATCC 43895 and Listeria monocytogenes ATCC 15313 were obtained from the Korean Collection for Type Cultures (KCTC, Seoul, Korea).


Synthesis of silver nanoparticles Silver nanoparticles were synthesized using organosolv lignin as reducing and capping

agent. The lignin was dried at 80 °C for 6 h before use. One hundred twenty milligrams of dried lignin was dissolved in 100 mL of chloroform with stirring. Since the organosolv lignin was not dissolved completely in chloroform, the solution was filtered through Whatman filter paper No. 1 to remove undissolved debris after 2 h,. The undissolved lignin was dried and weighed as around 40 mg. Two different amounts of silver nitrate (0.5 and 1.0 wt% of silver nitrate based on


PLA) were dissolved in ethanol and added dropwise to the lignin solution. The mixtures were kept stirring for 24 h. The change in color of the solution into yellow indicated the formation of silver nanoparticles.


Preparation of PLA/lignin/AgNPs composite films For the preparation of PLA/lignin/AgNPs composite films, 4 g PLA was added slowly

into the above prepared AgNPs solution and stirred continuously at 22±2 °C for 48 h. The fully solubilized film-forming solution was cast evenly onto a levelled Teflon film-coated glass plate (24 cm × 30 cm) and allowed to dry at room temperature (22±2 °C) for about 24 h. The dried films were peeled off from the plate and conditioned in a humidity chamber set at 25 °C and 50% RH for 48 h before further analysis. The films with neat PLA and PLA with lignin were prepared for comparison.


Characterization of PLA/lignin/AgNPs composite films

2.4.1. Surface morphology The surface morphology of the neat PLA, PLA/lignin, and PLA/lignin/AgNPs composite films was observed using a field emission scanning electron microscope (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan). The film samples were attached to the specimen holder, sputter coated with platinum, and the image was analyzed at an accelerating voltage of 3 kV.

2.4.2. Surface color and optical properties of composite films


The Hunter color values (L, a, and b) of the composite films were measured using a chroma meter (Minolta, CR-200, Tokyo, Japan) using a white color standard plate (L = 97.75, a = - 0.49, and b = 1.96) as a background. The total color difference (∆E) was calculated as follows:

where ΔL, Δa, and Δb are differences between each color value of the standard color plate and film specimen. Five measurements were taken for each film, and the average values are reported. The optical properties of the composite films were determined by measuring the absorption of light at 200-700 nm using a UV-visible spectrophotometer (Mecasys Optizen POP Series UV/Vis, Seoul, Korea). The UV-barrier property and transparency of the films was determined by measuring percent transmittance of light at 280 and 660 nm, respectively.

2.4.3. FTIR and XRD analysis Fourier transform infrared (FTIR) spectra of the composite films were measured by using an attenuated total reflectance-Fourier transform infrared (AT-FTIR) spectrophotometer (TENSOR 37 spectrophotometer with OPUS 6.0 software, Billerica, MA, USA). The spectra were recorded as 32 scans per samples at a resolution of 4 cm-1. X-ray diffraction (XRD) pattern of the films was analyzed by using an X-ray diffractometer (PANalytical X’ pert pro MRD diffractometer, Amsterdam, Netherlands). The film samples (2.5 × 2.5 cm) were placed on a glass slide and the spectra were recorded using Cu Kα radiation (wavelength of 0.1541 nm) and a nickel monochromator filtering wave at 40 kV and 30 mA. The diffraction pattern was recorded at 2θ = 30-80o with a scanning speed of 0.4°/min at room temperature (22±2 °C).


2.4.4. Thermogravimetric analysis Thermal stability of the composite films was determined using a thermogravimetric analyzer (Hi-Res TGA 2950, TA Instrument, New Castle, DE, USA). The film samples were taken in a standard aluminum pan and heated from 30 to 600 °C at the rate of 10 °C/min under a nitrogen flow of 50 mL/min. A derivative form of TGA (DTG) was obtained using differentials of TGA values which were calculated using a central finite difference method as follows [9]:

where Wt+Δt and Wt−Δt are the residual weight of the sample at time t+Δt and t-Δt, respectively, and Δt is the time interval for reading a residual sample weight. The maximum decomposition temperature (Tmax) of the composite films was obtained from DTG curve.

2.4.5. Mechanical properties The films were cut into 2.54 cm × 15 cm strips using a precision double blade cutter (model LB.02/A, Metrotec, S.A., San Sebastian, Spain) and the thickness of films was measured using a digital micrometer (Digimatic Micrometer, QuantuMike IP 65, Mitutoyo, Japan) with an accuracy of 0.001 mm. Five random locations around each film sample were measured and the average thickness was used for the calculation of tensile strength. The mechanical properties of the composite films in terms of tensile strength (TS), elongation at break (EAB), and elastic modulus (EM) were determined using an Instron Universal Testing Machine (Model 5565, Instron Engineering Corporation, Canton, MA, USA) in a tensile mode with an initial grip separation and a crosshead speed set at 50 mm and 50 mm/min, respectively [8]. The TS was calculated in MPa by dividing the maximum load (N) by the initial cross-sectional area (m2) of the film sample. The EAB was calculated in % by dividing 8

the extension at the rupture of the film by the initial length of the film (50 mm) and multiplying it by 100. The EM was determined in GPa from the slope of a linear portion of the stress-strain curve.

2.4.6. Water vapor permeability The water vapor permeability (WVP) of films was determined gravimetrically using water vapor transmission measuring cups by the ASTM E96-95 standard method with a modification [22]. The WVP measuring cup was filled with 18 mL of distilled water and the film sample (7.5 cm × 7.5 cm) was placed on the top of the cup and sealed tightly to prevent the leakage of water vapor. The assembled WVP cup was weighed and subsequently placed in a controlled environmental chamber set at 25 °C and 50% RH. Weight change of the cup was determined every 3 h for 24 h. The water vapor transmission rate (WVTR; g/m2.s) of the film was calculated by using the slope of the steady-state (linear) portion of weight loss versus time plot. Then, the WVP (g.m/m2.s.Pa) of the film was calculated as follows:

where L was the mean thickness of the film (m) and Δp was water vapor partial pressure difference (Pa) across the film, which was calculated by the method of Gennadios et al [22].

2.4.7. Antibacterial activity The antibacterial activity of composite films was examined against food-borne pathogenic Gram-positive bacteria, L. monocytogenes and Gram-negative bacteria, E. coli [8]. The test bacteria were aseptically inoculated in the BHI and TSB broth, respectively, and subsequently incubated at 37 °C for 16 h. The inoculum was diluted and 200 μL of diluted 9

inoculum (108-109 CFU/mL) was aseptically transferred to 50 mL of BHI and TSB broth containing 100 mg film samples and incubated at 37 °C for 15 h with mild shaking. The samples were taken out at 3, 6, 9, 12 and 15 h and inhibitory effect was estimated by measuring the total viable cell count after plating the samples on agar plates with serial dilution. Broth without film was used as a positive control. Antimicrobial tests were performed in triplicate with individually prepared films.


Statistical analysis Measurement of each property of the films was performed in triplicate with individually

prepared film samples as the replicated experimental units and the results were presented as mean values ± standard deviations (SD). One-way analysis of variance (ANOVA) was performed and the significance of each mean property value was determined (p < 0.05) with Duncan's multiple range tests using the SPSS statistical analysis computer program for Windows (SPSS Inc., Chicago, IL, USA).


Results and discussion


Synthesis of silver nanoparticles The filtrate containing lignin appeared slightly yellowish tint; however, the color was more

intensified to dark brown when silver nanoparticles were synthesized. The change in color of the solution indicated the formation of AgNPs due to the reduction of silver nitrate to metallic silver by lignin. The color intensity of the solution was dependent on the amount of silver nitrate used.


Lignin, a large and complex phenolic polymer, possesses aliphatic and aromatic hydroxyl groups, which have potential as reducing and capping agent during silver nanoparticle synthesis [23].


Characterization of PLA/lignin/AgNPs composite films

3.2.1. Surface morphology and optical properties All the films prepared were smooth, flexible, and free standing. The microstructure of the surface of the films was observed using a FE-SEM as shown in Fig. 1. The surface of the neat PLA and PLA/lignin composite films was smooth and compact. However, the surface of composite films incorporated with AgNPs was comparatively rough. Silver nanoparticles were spherical with the size less than 100 nm, and they were distributed uniformly on the surface of the composite films. Both nanocomposite films prepared with different silver nitrate concentration (0.5 and 1.0 wt%) exhibited a slightly aggregated form of AgNPs, however, the degree of aggregation was higher in the films added with the higher amount of silver nitrate. The aggregation might be due to a poor dispersion of AgNPs at high concentration in the system. Fig. 2 shows the light absorption spectra of the films in the wavelength of 200-700 nm. The neat PLA film was highly transparent, which did not show any light absorption at the wavelength above 240 nm. On the other hand, PLA film blended with lignin exhibited absorption in nearly the entire wavelength range of UV, which was mainly due to phenolic groups and conjugated carbonyl groups present in lignin [18]. The PLA films incorporated with silver nitrate exhibited additional peak with the absorption peak around 450 nm, which was attributed to the surface plasmonic resonance of AgNPs in the composite films [7,24]. Though the position and intensity of AgNPs peak in both nanocomposite films (PLA/lignin/AgNP0.5 and PLA/lignin/AgNP1.0)


were similar, however, the film with a higher concentration of silver nitrate showed the broader peak. This broadening of peak might be due to the aggregation of AgNPs at higher concentration of AgNPs. Apparently, the neat PLA film was transparent without any color tint, but the film turned to yellow after blending with lignin. On the other hand, the color of the PLA film incorporated with AgNPs became yellowish brown. The intensity of brown color increased with the increase in the concentration of silver nitrate, which also indicated indirectly the amount of AgNPs formed was dependent on the concentration of silver nitrate. The Hunter L-value (lightness) of the neat PLA film was 93.3±7.4, which decreased significantly (p

silver nanoparticles composite films with UV light barrier and antibacterial properties.

Organosolv lignin was used as a reducing agent for the preparation of silver nanoparticles (AgNPs) and their incorporation into poly(lactide) (PLA) po...
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