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Improving properties of Hanji by coating chitosan–silver nanoparticle solution
Accepted Manuscript Title: Improving properties of Hanji by coating chitosan–silver nanoparticle solution Author: Jeyoung Jung Gownolla Malegowd Raghavendra Dowan Kim Jongchul Seo PII: DOI: Reference:
S0141-8130(16)30678-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.09.067 BIOMAC 6534
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-6-2016 10-9-2016 19-9-2016
Please cite this article as: Jeyoung Jung, Gownolla Malegowd Raghavendra, Dowan Kim, Jongchul Seo, Improving properties of Hanji by coating chitosan–silver nanoparticle solution, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.09.067 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.
Improving properties of Hanji by coating chitosan–silver nanoparticle solution Jeyoung Jung, Gownolla Malegowd Raghavendra, Dowan Kim, Jongchul Seo* Department of Packaging, Yonsei University, Gangwondo 220-710, Republic of Korea *Corresponding author: email: [email protected]
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Highlights Hanji (Korean traditional paper) was coated with chitosan silver nanoparticle solutions The properties of the Hanji were investigated as a function of the dilution ratio Undiluted, 1/10, 1/100 and 1/1000 dilutions were employed. The maximum level of dilution that influences the properties of Hanji was determined
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Abstract A chitosan–silver nanoparticle solution (CSNS) was applied as a coating material to Hanji (Korean traditional paper), and the properties of the coated paper were investigated as a function of the dilution ratio. The required CSNS was first prepared from AgNO3 (30 mmol) by utilizing chitosan as a reducing and stabilizing agent via ultrasonication. The as-prepared CSNS was diluted to various ratios (undiluted, 1/10, 1/100, and 1/1000) and applied to Hanji by a dip-coating method. The tensile, burst, oil resistance, and antibacterial properties of the coated Hanji against Escherichia coli were evaluated. Among the various dilution ratios, the maximum level of dilution that can positively influence the tensile, burst, oil resistance, and antibacterial properties of Hanji was identified as 1/10, 1/100, 1/10 and 1/1000 of the pure CSNS, respectively. These findings are significant because a specific property of Hanji can be economically improved by changing the dilution ratio. Keywords: Hanji; Silver Nanoparticles; Chitosan
1. Introduction Hanji, the Korean traditional paper, is considered to be one of the most stable and durable papers in the world. It was estimated that the life span of Korean paper exceeds 1,000 years [1]. This superiority of Korean paper comes from the material of which it is made. It is 3
traditionally made of bast fibers obtained from one-year-old paper mulberry trees [2]. The cellulose found in the pulp of the paper mulberry has a very high average degree of polymerization (in the 7000–9000 range) and exhibits a high molar mass that is comparable to that of the celluloses produced by bacteria or tunicates [2]. Currently, Hanji is available on the market with various percentages of mulberry and kraft pulp. As a packaging material, Hanji is used as parcel wrappers, food packing, and a traditional covering material. However, Hanji possesses poor barrier, mechanical, and antimicrobial properties. This limits its effective use in industry. Because Hanji can be considered to be a system of long cellulose fibers and pores, its properties, such as its mechanical strength, oil resistance, and antimicrobial activity, can be expected to be modified by surface treatment such as coating, calendaring, and corona discharge [3, 4]. In the paper industry, chitosan has been used as an additive for surface treatment. Chitosan is readily compatible with paper; hence, it enhances the mechanical and barrier properties of paper when applied as a coating. In addition, chitosan possesses antimicrobial properties resulting from the cationic characteristics of the amino groups in repeating units [5, 6]. Owing to these significant properties, chitosan has become one of the most interesting materials for paper coatings [7]. The addition of chitosan with unbleached sulfite reportedly enhanced the burst, dry-tensile, and wet-tensile properties of papers [8]. Chitosan-grafted copolymers have been exploited for making paper products with improved dry strength [9]. Further, to overcome the poor oil resistance, the paper substrate is generally coated with oilresistant coatings. Chitosan was reported to function as an oil-resistant coating material [10, 11]. In recent years, silver nanoparticles (AgNPs) have received increased attention as potential antimicrobial agents [12-14]. This material kills a broad spectrum of microorganisms including multidrug-resistant bacteria [15]. Using AgNPs, Liu et al. developed a multipurpose antibacterial paper [16]. The use of AgNPs for food packaging applications has been reported by various authors and has been reviewed recently by Carbone et al. [17-19]. Fernández et al. developed bactericidal water filters based on biosynthesized AgNPs and applied them to water purification [20]. The better adhesion properties of chitosan than AgNPs towards cellulose substrate, and the better antimicrobial properties of AgNPs than chitosan, can make the CSNS coated 4
Hanji to overcome the inherent deficiencies that would generate by coating either chitosan or AgNPs alone [21, 22]. Hence, in this study, a chitosan–silver nanoparticle solution (CSNS) was formulated and applied to Hanji. The required CSNS was formulated via ultrasonication. The ultrasonication technique was employed because of its close relevance to synthesis of various nanomaterials [23-25]. To find the highest possible level of dilution that improves the intended property of Hanji for a specific application, the as-prepared CSNS was diluted to 1/10, 1/100 and 1/1000 of the original concentration and applied to Hanji by a dip-coating method. The effect of coating on the mechanical, oil resistance, and antibacterial properties was investigated. These findings are expected to provide economic benefits and expand the applications of Hanji in various industrial fields.
2. Experimental 2.1. Materials Handmade Hanji manufactured traditionally from 20% mulberry fiber with kraft pulp was obtained from a local company, Wonju, South Korea. Chitosan (DD: 75%–85%) of medium molecular weight (Mv = 190–310 kDa) with a Brookfield viscosity of 200–800 cP (CAS 9012-76-4) and silver nitrate (AgNO3) (ACS reagent, ≥99.0%) were purchased from Sigma Aldrich. 10% acetic acid solution was purchased from Duksan Pure Chemical Co. Ltd., Korea. All chemicals were used as received without further purification. Deionized water was used throughout the experiments. 2.2. Preparation of chitosan–silver nanoparticle solution (CSNS) First, chitosan solution (1.5 wt.%) was prepared from 2.0 v/v% acetic acid solution (prepared from 10 % acetic acid) by vigorous stirring over a heating magnetic stirrer at 90 C for 8 h. The obtained chitosan solution was cooled to room temperature. Subsequently, AgNO3 (30 mmol) previously diluted with 5 mL of distilled water, was added to the chitosan solution and stirred for 5 min to obtain a chitosan–AgNO3 solution. The solution was transferred to a beaker and then irradiated to ultrasonication for 60 min, with a probe temperature of 90 C. At the end of the reaction, the color of the solution changed from 5
colorless to ruby red, indicating that AgNPs had formed, and hence, that a CSNS was obtained. The prepared CSNS was allowed to cool to room temperature and used for coating Hanji in the subsequent steps. 2.3. Coating of CSNS on Hanji The as-prepared CSNS was diluted to 1/10, 1/100, and 1/1000 of the original concentration by adding deionized water. A dip-coating technique was employed to coat these solutions on Hanji. Hanji papers were dipped individually into the undiluted and the diluted coating solutions for 30 s to obtain various CSNS-coated Hanji samples. The excess solution was removed by gently press-passing the coated Hanji samples between two glass rods. Next, the coated Hanji samples were dried in an oven at 90 C for 10 min and were stored protected from light in air-tight polyethylene covers. The Hanji samples coated with the undiluted, 1/10, 1/100, and 1/1000 diluted CSNS are denoted as SA, SB, SC, and SD, respectively. The preparation of ultrasonicated chitosan–silver nanoparticle solution and its application to Hanji was shown in Scheme 1. 3. Characterization 3.1. UV–vis analysis The UV–vis spectrum of the CSNS was recorded in the 300–700 nm range using a UV–vis spectrophotometer (JASCO V-650, JASCO International Co., Ltd., Hachioji, Japan). 3.2. Fourier transform infrared (FTIR) analysis To understand the functional group interaction during AgNP formation, FTIR spectra were recorded using a Spectrum 65 FTIR spectrometer (PerkinElmer Co., Ltd., Massachusetts, USA) in the range of 4000–400 cm−1. 3.3. Transmission electron microscopy (TEM) analysis The morphology of AgNPs was studied using a transmission electron microscope (Tecnai G2 Spirit, FEI Co., Ltd., Oregon, USA) operating at an accelerating voltage of 120 kV. The TEM samples were prepared by diluting the as-prepared CSNS and dropping the 6
diluted solutions onto carbon-coated copper grids (400 mesh). 3.4. Scanning electron microscopy/energy-dispersive spectroscopy (SEM/EDS) The surface morphologies of the Hanji samples were analyzed using a scanning electron microscope equipped with an energy-dispersive X-ray spectrometer (Quanta FEG250, FEI Co., Ltd., Oregon, USA). Before the analysis, the samples were coated with platinum for 60 s in a vacuum chamber. 3.5. Tensile and burst strength The tensile strength of the samples was evaluated using a universal testing machine (QM100T, Qmesys Co., Gun-po, Korea) according to ASTM D828-97. The burst strength of the samples was measured using a burst test machine (Model BT-1000, TM Electronics Inc., Massachusetts, USA) according to ASTM F1140. For both tests, eight measurements were evaluated for each composition. 3.6. Oil resistance The oil resistance of the samples was evaluated according to TAPPI standard test T559 pm-96 using a Kit test procedure [10]. The samples were tested using a series of solutions with different Kit numbers (1–12) containing specific proportions of three reagents: castor oil, toluene, and n-heptane. Various solutions were dropped onto the sample surface. After 15 s, the oils were removed with a tissue. The highest-numbered liquid that remained on the surface of the sample without causing staining was reported as the Kit number for the sample. The lower the Kit number was, the less aggressive the oil was for the sample (i.e., the higher the surface energy was), and vice versa. 3.7. Antibacterial activity The antibacterial activity of all the samples was tested using the inhibition zone and the JIS Z 2801 standard methods [26-28]. DH5α Escherichia coli (E. coli) was chosen as model bacteria. The required nutrient agar media for E. coli was prepared from MacConkey [29]. The agar media were sterilized in conical flasks at a pressure of 103.4 × 103 Pa for 15 min at 121 C. The sterilized media were then transferred to sterilized petri dishes on a clean 7
bench. After solidification of the growth media, bacterial cultures (50 µL) were inoculated by uniformly streaking the solidified agar surface. The sample discs were distributed and incubated for 24 h at 38 C in an incubation chamber. Next, the inhibition zones that formed around the samples were measured. For JIS Z 2801 standard method, a single colony of the freshly grown bacteria was transferred into a 10 mL aliquot of nutrient broth to obtain a fresh E. coli culture (at 38 °C for 24 h). Then, 1 mL of the freshly cultured E. coli was inoculated into the sample and a thin sterile film was covered. The complete sample set along with E. coli was then transferred to a 10 mL aliquot of broth and allowed at 38 °C for 24 h. Under these conditions, the growth of E. coli was influenced by the sample. The sample-influenced E. coli culture was serially diluted and transferred over MacConkey agar plates. Finally, the bacterial count i.e., number of colony forming units per mL (CFU/mL) was determined by incubating these plates (90% RH and 38 ± 1 °C for 24 h). The antimicrobial rate, R was calculated using the equation (2): R(%) =
(B − C) X 100 − − − − − −(2) B
Where, B is the number of CFU/mL for pure Hanji and C is the number of CFU/mL for the CSNS coated Hanji samples. The test was performed in triplicate sets. 4. Results and discussion 4.1. Analysis of AgNPs in chitosan solution UV-vis spectroscopy is one of the most important techniques for investigating the formation of AgNPs [27]. Chitosan has electron-donating groups such as hydroxyl and amino groups [21]. When AgNO3 was added to the chitosan solution, silver ions would become attached to the chitosan molecules by electrostatic interaction with these groups. With time, under the influence of ultrasonication, the silver ions were reduced to AgNPs. AgNPs formation can be identified through a localized surface plasmon resonance band in the spectra at 435 nm [30, 31]. In Figure 1(a), the appearance of a UV-vis absorbance peak at 435 nm for the CSNS indicates the formation of AgNPs. The morphology of the AgNPs was revealed by TEM [Figure 1(b)]. The formed 8
AgNPs are clearly almost spherical in shape with sizes below 30 nm. Further, aggregation was not found, indicating that the AgNPs might be stabilized by the polymeric chitosan chains. Similar observation was reported in the literature [32]. To investigate the functional groups involved in the formation of AgNPs, FTIR spectroscopy was performed for pure chitosan and the CSNS, as shown in Figure 1(c). Pure chitosan exhibited characteristic peaks at 3000–3500 cm−1, which are assigned to the overlapped –NH and –OH stretching vibrations [33]. The peaks of C=O stretching, N–H bending, and O–H bending vibration were also observed at 1651, 1558, and 1410 cm−1, respectively [21]. As expected, the CSNS also exhibited characteristic peaks similar to those of pure chitosan. However, the N–H bending peak at about 1550 cm−1 was shifted to 1535 cm−1. In addition, a new band at about 1690 cm−1, corresponding to carbonyl stretching vibrations, was obtained. The presence of the band at 1690 cm–1 may indicate that reduction of the silver ions is coupled to oxidation of the hydroxyl groups in the chitosan molecule [4]. This suggests the interaction of silver with electronegative nitrogen and oxygen atoms. Thus, the AgNPs can be expected to be capped up (stabilized) by the polymeric chitosan chains, resulting in no aggregation of the formed AgNPs [as evidenced by TEM, Figure 1(b)]. The UV-vis, TEM, and FTIR results indicate that a CSNS with nanosized AgNPs stabilized by polymeric chitosan chains was successfully prepared using ultrasonication system. The obtained CSNS was applied to improve the properties of Hanji, specifically, the mechanical properties, oil resistance, and antibacterial properties. 4.2. Morphological analysis of CSNS-coated Hanji To examine the coating of chitosan–AgNPs on Hanji, SEM observations were performed for neat Hanji and coated Hanji (SA and SD) samples, as shown in Figure 2. The SEM image of the neat Hanji showed a mottled and rough appearance due to interwoven fibers. In contrast, SA showed significantly distinguishable morphological changes compared to neat Hanji. SA showed a rather smooth surface, indicating a clear chitosan–AgNP coating over the Hanji substrate. This may be the result of AgNP aggregation during coating and drying [34]. Furthermore, the SD sample exhibited decreased coverage of the mottled and rough appearance of interwoven fibers owing to dilution of the CSNS coating. It can be 9
predicted that because of the dilute coating, SD exhibited a smoother surface than neat Hanji. The observed phenomenon may be due to removal of weakly bound cellulosic impurities and debris during dilute coating over the Hanji substrate. EDS spectra were recorded to examine the presence of elemental silver over the samples. The neat Hanji did not show the characteristic silver peak at 3 keV, whereas the coated Hanji samples did. As expected, sample SD with the most highly diluted CSNS showed a lower-intensity silver peak than SA [27]. 4.3. Effect of CSNS coating on Hanji 4.3.1. Mechanical properties As shown in Figure 3, neat Hanji showed relatively poor mechanical properties (tensile and burst strength) compared to undiluted-CSNS-coated Hanji (SA). However, Hanji coated with the diluted CSNS showed a decreasing trend, where the decrease was proportional to the level of dilution. SA's highest mechanical strength is attributed to the CSNS coating. The mechanical properties of paper depend strongly on the internal interaction among cellulose fibers themselves and the reinforcement effect of the coating layer [35]. As the interaction of cellulose fibers is common to all the Hanji samples, the observed high value of the mechanical strength for SA is most likely due to the effect of the coating. Chitosan can reportedly form a tough, flexible coating layer with good tear resistance that exhibits improvement in all of the mechanical properties [36]. The chemical similarity of chitosan to the cellulose in Hanji is assumed to facilitate the formation of strong internal hydrogen bonds and produce a tough compatible fiber–binder system in the coated Hanji [22]. However, upon dilution, the fiber–binder system is weakened by the quantitative decrease in internal hydrogen bonds. Hence, the decreasing trend in the mechanical properties was observed with increasing dilution ratio and the resulting loss of already existing interfiber bonding networks in Hanji. 4.3.2. Oil resistance The Kit numbers of the samples, which represent the level of oil resistance, are shown in Figure 4. SC and SD showed the same Kit numbers as neat Hanji. However, 10
samples SA and SB showed increased Kit numbers; in particular, SA showed the highest Kit number. The obtained results are quite expected. From the data, it is possible to state that the CSNS coating improved the oil resistance of Hanji up to a dilution level of 1/10. The improved oil resistance of SA and SB compared to that of neat Hanji results from the CSNS coating, which forms a layer over the surface of Hanji and resists the passage of oil through it [35]. In addition, the CSNS that penetrated the internal voids of Hanji in the coating process would exhibit a positive effect by forming sufficient interfiber bonds with the cellulose in Hanji. These interfiber bonds can bridge the interfiber distances of the cellulose fiber surfaces and establish new bonds between the fibers and fibrils. Thus, a network-like system is created that would not have otherwise formed before CSNS coating; consequently, a physical barrier against oil penetration is developed and oil spread is arrested [22]. The lower oil resistance of SB compared to that of SA can be attributed to coating with the diluted CSNS, which quantitatively reduces the formation of additional new bonds and produces a weaker network than that in SA. Further, owing to the relatively high dilution, no further improvement in the oil barrier properties beyond neat Hanji is observed in SC and SD. 4.3.3. Antibacterial properties The inhibitory effect of neat Hanji and the coated Hanji samples (SA, SB, SC, and SD) is shown in Figure 5. As expected, no inhibition zone was observed for neat Hanji. However, SA and SB showed a clear inhibition zone of over 1 mm against E. coli. Compounds showing an inhibition zone of >1 mm are commonly considered good antibacterial agents according to the Swiss Norm (SN) 195920-ASTM E 2149-01[37]. Hence, the SA and SB samples obtained using the current approach can be considered good antibacterial agents. Further, to understand the positive influence of CSNS in imparting the antibacterial property to the Hanji, JIS Z 2801 standard was performed and the results are also shown in Figure 5. From the data, it is clear that irrespective of dilution all the coated samples showed higher antibacterial activity than neat Hanji (SA > SB > SC > SD> neat Hanji). This trend was expected, since it follows the trend of decreasing distribution of AgNPs with increasing dilution ratio, as confirmed by SEM and EDS. It is apparent that a stoichiometrically smaller population of AgNPs enables less significant interaction with bacterial colonies, resulting in decreased reduction in bacterial colonies with dilution. It is 11
also clear that, though neat Hanji and SD showed zero inhibition, yet the R (%) data, exhibited a clear difference, which showed 93.4 % higher antibacterial activity for SE than neat Hanji. This was evidently due to the effect of CSNS coating which could able to reduce bacterial growth effectively than neat Hanji. The antibacterial action is evidently due to CSNS coating, which is a combination of two antibacterial components, AgNPs and chitosan. The antibacterial activity of AgNPs is well known. Though the mechanism of the inhibitory effects of AgNPs on microorganisms is not completely clear, yet it could devastate causing leakage of lipopolysaccharide molecules and membrane proteins, leading to bacteria’s death [11]. The antibacterial action of chitosan is due to presence of charged groups in its backbone. These charged groups exhibit interactions with bacterial wall and results in the hydrolysis of peptidoglycans of microorganism wall and triggers the leakage of intracellular electrolytes, leading to the death of microorganism [38]. 5. Conclusion The functional properties of Hanji coated with a CSNS were investigated as a function of the dilution ratio. In the current approach, the CSNS was successfully prepared from AgNO3 and chitosan via ultrasonication and then applied to Hanji by a dip-coating method. UV-vis and TEM revealed the presence of AgNPs in the CSNS. FTIR spectroscopy indicated that the AgNPs were formed by the –OH groups of chitosan. SEM/EDS confirmed that the degree to which the CSNS coated the Hanji was in accordance with the dilution ratio. Improved tensile strength was observed for Hanji coated with the CSNS up to a dilution level of 1/10. In addition, improved burst strength was obtained by coating the CSNS up to a dilution level of 1/100. Further, it was found that a CSNS coating diluted by up to 1/10 of its original concentration can improve the oil resistance of Hanji. However, the Hanji exhibited improved antibacterial properties after it was coated with the CSNS even up to a dilution level of 1/1000. On the basis of the obtained results, it is possible to conclude that specific properties of Hanji can be improved by changing the dilution ratio. Further, among the four dilution ratios: undiluted, 1/10, 1/100 and 1/1000, ‘1/10’ can be considered as the ‘highest common dilution ratio’ that can improve all the four functional properties of Hanji. These significant findings have numerous versatile applications in Hanji-based packaging industries. 12
Acknowledgments This work was supported by the Korea Institute of Planning and Evaluation for Technology in the Ministry for Food, Agriculture, Forestry, and Fisheries of the Korean Government (No. IPET314050-3).
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Caption for Figures Scheme 1: Preparation of ultrasonicated chitosan–silver nanoparticle solution (CSNS) and its 17
application to Hanji Figure 1: a) UV-Vis spectrum of CSNS; b) TEM image of silver nanoparticles in CSNS; c) FTIR spectra of chitosan solution and CSNS Figure 2: SEM and EDS of neat Hanji, SA and SD Figure 3: Tensile and burst strength of neat Hanji, SA, SB, SC and SD Figure 4: Oil resistance of neat Hanji, SA, SB, SC and SD Figure 5: Antibacterial activity of neat Hanji, SA, SB, SC and SD
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S0141-8130(16)30678-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.09.067 BIOMAC 6534
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-6-2016 10-9-2016 19-9-2016
Please cite this article as: Jeyoung Jung, Gownolla Malegowd Raghavendra, Dowan Kim, Jongchul Seo, Improving properties of Hanji by coating chitosan–silver nanoparticle solution, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.09.067 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.
Improving properties of Hanji by coating chitosan–silver nanoparticle solution Jeyoung Jung, Gownolla Malegowd Raghavendra, Dowan Kim, Jongchul Seo* Department of Packaging, Yonsei University, Gangwondo 220-710, Republic of Korea *Corresponding author: email: [email protected]
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Highlights Hanji (Korean traditional paper) was coated with chitosan silver nanoparticle solutions The properties of the Hanji were investigated as a function of the dilution ratio Undiluted, 1/10, 1/100 and 1/1000 dilutions were employed. The maximum level of dilution that influences the properties of Hanji was determined
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Abstract A chitosan–silver nanoparticle solution (CSNS) was applied as a coating material to Hanji (Korean traditional paper), and the properties of the coated paper were investigated as a function of the dilution ratio. The required CSNS was first prepared from AgNO3 (30 mmol) by utilizing chitosan as a reducing and stabilizing agent via ultrasonication. The as-prepared CSNS was diluted to various ratios (undiluted, 1/10, 1/100, and 1/1000) and applied to Hanji by a dip-coating method. The tensile, burst, oil resistance, and antibacterial properties of the coated Hanji against Escherichia coli were evaluated. Among the various dilution ratios, the maximum level of dilution that can positively influence the tensile, burst, oil resistance, and antibacterial properties of Hanji was identified as 1/10, 1/100, 1/10 and 1/1000 of the pure CSNS, respectively. These findings are significant because a specific property of Hanji can be economically improved by changing the dilution ratio. Keywords: Hanji; Silver Nanoparticles; Chitosan
1. Introduction Hanji, the Korean traditional paper, is considered to be one of the most stable and durable papers in the world. It was estimated that the life span of Korean paper exceeds 1,000 years [1]. This superiority of Korean paper comes from the material of which it is made. It is 3
traditionally made of bast fibers obtained from one-year-old paper mulberry trees [2]. The cellulose found in the pulp of the paper mulberry has a very high average degree of polymerization (in the 7000–9000 range) and exhibits a high molar mass that is comparable to that of the celluloses produced by bacteria or tunicates [2]. Currently, Hanji is available on the market with various percentages of mulberry and kraft pulp. As a packaging material, Hanji is used as parcel wrappers, food packing, and a traditional covering material. However, Hanji possesses poor barrier, mechanical, and antimicrobial properties. This limits its effective use in industry. Because Hanji can be considered to be a system of long cellulose fibers and pores, its properties, such as its mechanical strength, oil resistance, and antimicrobial activity, can be expected to be modified by surface treatment such as coating, calendaring, and corona discharge [3, 4]. In the paper industry, chitosan has been used as an additive for surface treatment. Chitosan is readily compatible with paper; hence, it enhances the mechanical and barrier properties of paper when applied as a coating. In addition, chitosan possesses antimicrobial properties resulting from the cationic characteristics of the amino groups in repeating units [5, 6]. Owing to these significant properties, chitosan has become one of the most interesting materials for paper coatings [7]. The addition of chitosan with unbleached sulfite reportedly enhanced the burst, dry-tensile, and wet-tensile properties of papers [8]. Chitosan-grafted copolymers have been exploited for making paper products with improved dry strength [9]. Further, to overcome the poor oil resistance, the paper substrate is generally coated with oilresistant coatings. Chitosan was reported to function as an oil-resistant coating material [10, 11]. In recent years, silver nanoparticles (AgNPs) have received increased attention as potential antimicrobial agents [12-14]. This material kills a broad spectrum of microorganisms including multidrug-resistant bacteria [15]. Using AgNPs, Liu et al. developed a multipurpose antibacterial paper [16]. The use of AgNPs for food packaging applications has been reported by various authors and has been reviewed recently by Carbone et al. [17-19]. Fernández et al. developed bactericidal water filters based on biosynthesized AgNPs and applied them to water purification [20]. The better adhesion properties of chitosan than AgNPs towards cellulose substrate, and the better antimicrobial properties of AgNPs than chitosan, can make the CSNS coated 4
Hanji to overcome the inherent deficiencies that would generate by coating either chitosan or AgNPs alone [21, 22]. Hence, in this study, a chitosan–silver nanoparticle solution (CSNS) was formulated and applied to Hanji. The required CSNS was formulated via ultrasonication. The ultrasonication technique was employed because of its close relevance to synthesis of various nanomaterials [23-25]. To find the highest possible level of dilution that improves the intended property of Hanji for a specific application, the as-prepared CSNS was diluted to 1/10, 1/100 and 1/1000 of the original concentration and applied to Hanji by a dip-coating method. The effect of coating on the mechanical, oil resistance, and antibacterial properties was investigated. These findings are expected to provide economic benefits and expand the applications of Hanji in various industrial fields.
2. Experimental 2.1. Materials Handmade Hanji manufactured traditionally from 20% mulberry fiber with kraft pulp was obtained from a local company, Wonju, South Korea. Chitosan (DD: 75%–85%) of medium molecular weight (Mv = 190–310 kDa) with a Brookfield viscosity of 200–800 cP (CAS 9012-76-4) and silver nitrate (AgNO3) (ACS reagent, ≥99.0%) were purchased from Sigma Aldrich. 10% acetic acid solution was purchased from Duksan Pure Chemical Co. Ltd., Korea. All chemicals were used as received without further purification. Deionized water was used throughout the experiments. 2.2. Preparation of chitosan–silver nanoparticle solution (CSNS) First, chitosan solution (1.5 wt.%) was prepared from 2.0 v/v% acetic acid solution (prepared from 10 % acetic acid) by vigorous stirring over a heating magnetic stirrer at 90 C for 8 h. The obtained chitosan solution was cooled to room temperature. Subsequently, AgNO3 (30 mmol) previously diluted with 5 mL of distilled water, was added to the chitosan solution and stirred for 5 min to obtain a chitosan–AgNO3 solution. The solution was transferred to a beaker and then irradiated to ultrasonication for 60 min, with a probe temperature of 90 C. At the end of the reaction, the color of the solution changed from 5
colorless to ruby red, indicating that AgNPs had formed, and hence, that a CSNS was obtained. The prepared CSNS was allowed to cool to room temperature and used for coating Hanji in the subsequent steps. 2.3. Coating of CSNS on Hanji The as-prepared CSNS was diluted to 1/10, 1/100, and 1/1000 of the original concentration by adding deionized water. A dip-coating technique was employed to coat these solutions on Hanji. Hanji papers were dipped individually into the undiluted and the diluted coating solutions for 30 s to obtain various CSNS-coated Hanji samples. The excess solution was removed by gently press-passing the coated Hanji samples between two glass rods. Next, the coated Hanji samples were dried in an oven at 90 C for 10 min and were stored protected from light in air-tight polyethylene covers. The Hanji samples coated with the undiluted, 1/10, 1/100, and 1/1000 diluted CSNS are denoted as SA, SB, SC, and SD, respectively. The preparation of ultrasonicated chitosan–silver nanoparticle solution and its application to Hanji was shown in Scheme 1. 3. Characterization 3.1. UV–vis analysis The UV–vis spectrum of the CSNS was recorded in the 300–700 nm range using a UV–vis spectrophotometer (JASCO V-650, JASCO International Co., Ltd., Hachioji, Japan). 3.2. Fourier transform infrared (FTIR) analysis To understand the functional group interaction during AgNP formation, FTIR spectra were recorded using a Spectrum 65 FTIR spectrometer (PerkinElmer Co., Ltd., Massachusetts, USA) in the range of 4000–400 cm−1. 3.3. Transmission electron microscopy (TEM) analysis The morphology of AgNPs was studied using a transmission electron microscope (Tecnai G2 Spirit, FEI Co., Ltd., Oregon, USA) operating at an accelerating voltage of 120 kV. The TEM samples were prepared by diluting the as-prepared CSNS and dropping the 6
diluted solutions onto carbon-coated copper grids (400 mesh). 3.4. Scanning electron microscopy/energy-dispersive spectroscopy (SEM/EDS) The surface morphologies of the Hanji samples were analyzed using a scanning electron microscope equipped with an energy-dispersive X-ray spectrometer (Quanta FEG250, FEI Co., Ltd., Oregon, USA). Before the analysis, the samples were coated with platinum for 60 s in a vacuum chamber. 3.5. Tensile and burst strength The tensile strength of the samples was evaluated using a universal testing machine (QM100T, Qmesys Co., Gun-po, Korea) according to ASTM D828-97. The burst strength of the samples was measured using a burst test machine (Model BT-1000, TM Electronics Inc., Massachusetts, USA) according to ASTM F1140. For both tests, eight measurements were evaluated for each composition. 3.6. Oil resistance The oil resistance of the samples was evaluated according to TAPPI standard test T559 pm-96 using a Kit test procedure [10]. The samples were tested using a series of solutions with different Kit numbers (1–12) containing specific proportions of three reagents: castor oil, toluene, and n-heptane. Various solutions were dropped onto the sample surface. After 15 s, the oils were removed with a tissue. The highest-numbered liquid that remained on the surface of the sample without causing staining was reported as the Kit number for the sample. The lower the Kit number was, the less aggressive the oil was for the sample (i.e., the higher the surface energy was), and vice versa. 3.7. Antibacterial activity The antibacterial activity of all the samples was tested using the inhibition zone and the JIS Z 2801 standard methods [26-28]. DH5α Escherichia coli (E. coli) was chosen as model bacteria. The required nutrient agar media for E. coli was prepared from MacConkey [29]. The agar media were sterilized in conical flasks at a pressure of 103.4 × 103 Pa for 15 min at 121 C. The sterilized media were then transferred to sterilized petri dishes on a clean 7
bench. After solidification of the growth media, bacterial cultures (50 µL) were inoculated by uniformly streaking the solidified agar surface. The sample discs were distributed and incubated for 24 h at 38 C in an incubation chamber. Next, the inhibition zones that formed around the samples were measured. For JIS Z 2801 standard method, a single colony of the freshly grown bacteria was transferred into a 10 mL aliquot of nutrient broth to obtain a fresh E. coli culture (at 38 °C for 24 h). Then, 1 mL of the freshly cultured E. coli was inoculated into the sample and a thin sterile film was covered. The complete sample set along with E. coli was then transferred to a 10 mL aliquot of broth and allowed at 38 °C for 24 h. Under these conditions, the growth of E. coli was influenced by the sample. The sample-influenced E. coli culture was serially diluted and transferred over MacConkey agar plates. Finally, the bacterial count i.e., number of colony forming units per mL (CFU/mL) was determined by incubating these plates (90% RH and 38 ± 1 °C for 24 h). The antimicrobial rate, R was calculated using the equation (2): R(%) =
(B − C) X 100 − − − − − −(2) B
Where, B is the number of CFU/mL for pure Hanji and C is the number of CFU/mL for the CSNS coated Hanji samples. The test was performed in triplicate sets. 4. Results and discussion 4.1. Analysis of AgNPs in chitosan solution UV-vis spectroscopy is one of the most important techniques for investigating the formation of AgNPs [27]. Chitosan has electron-donating groups such as hydroxyl and amino groups [21]. When AgNO3 was added to the chitosan solution, silver ions would become attached to the chitosan molecules by electrostatic interaction with these groups. With time, under the influence of ultrasonication, the silver ions were reduced to AgNPs. AgNPs formation can be identified through a localized surface plasmon resonance band in the spectra at 435 nm [30, 31]. In Figure 1(a), the appearance of a UV-vis absorbance peak at 435 nm for the CSNS indicates the formation of AgNPs. The morphology of the AgNPs was revealed by TEM [Figure 1(b)]. The formed 8
AgNPs are clearly almost spherical in shape with sizes below 30 nm. Further, aggregation was not found, indicating that the AgNPs might be stabilized by the polymeric chitosan chains. Similar observation was reported in the literature [32]. To investigate the functional groups involved in the formation of AgNPs, FTIR spectroscopy was performed for pure chitosan and the CSNS, as shown in Figure 1(c). Pure chitosan exhibited characteristic peaks at 3000–3500 cm−1, which are assigned to the overlapped –NH and –OH stretching vibrations [33]. The peaks of C=O stretching, N–H bending, and O–H bending vibration were also observed at 1651, 1558, and 1410 cm−1, respectively [21]. As expected, the CSNS also exhibited characteristic peaks similar to those of pure chitosan. However, the N–H bending peak at about 1550 cm−1 was shifted to 1535 cm−1. In addition, a new band at about 1690 cm−1, corresponding to carbonyl stretching vibrations, was obtained. The presence of the band at 1690 cm–1 may indicate that reduction of the silver ions is coupled to oxidation of the hydroxyl groups in the chitosan molecule [4]. This suggests the interaction of silver with electronegative nitrogen and oxygen atoms. Thus, the AgNPs can be expected to be capped up (stabilized) by the polymeric chitosan chains, resulting in no aggregation of the formed AgNPs [as evidenced by TEM, Figure 1(b)]. The UV-vis, TEM, and FTIR results indicate that a CSNS with nanosized AgNPs stabilized by polymeric chitosan chains was successfully prepared using ultrasonication system. The obtained CSNS was applied to improve the properties of Hanji, specifically, the mechanical properties, oil resistance, and antibacterial properties. 4.2. Morphological analysis of CSNS-coated Hanji To examine the coating of chitosan–AgNPs on Hanji, SEM observations were performed for neat Hanji and coated Hanji (SA and SD) samples, as shown in Figure 2. The SEM image of the neat Hanji showed a mottled and rough appearance due to interwoven fibers. In contrast, SA showed significantly distinguishable morphological changes compared to neat Hanji. SA showed a rather smooth surface, indicating a clear chitosan–AgNP coating over the Hanji substrate. This may be the result of AgNP aggregation during coating and drying [34]. Furthermore, the SD sample exhibited decreased coverage of the mottled and rough appearance of interwoven fibers owing to dilution of the CSNS coating. It can be 9
predicted that because of the dilute coating, SD exhibited a smoother surface than neat Hanji. The observed phenomenon may be due to removal of weakly bound cellulosic impurities and debris during dilute coating over the Hanji substrate. EDS spectra were recorded to examine the presence of elemental silver over the samples. The neat Hanji did not show the characteristic silver peak at 3 keV, whereas the coated Hanji samples did. As expected, sample SD with the most highly diluted CSNS showed a lower-intensity silver peak than SA [27]. 4.3. Effect of CSNS coating on Hanji 4.3.1. Mechanical properties As shown in Figure 3, neat Hanji showed relatively poor mechanical properties (tensile and burst strength) compared to undiluted-CSNS-coated Hanji (SA). However, Hanji coated with the diluted CSNS showed a decreasing trend, where the decrease was proportional to the level of dilution. SA's highest mechanical strength is attributed to the CSNS coating. The mechanical properties of paper depend strongly on the internal interaction among cellulose fibers themselves and the reinforcement effect of the coating layer [35]. As the interaction of cellulose fibers is common to all the Hanji samples, the observed high value of the mechanical strength for SA is most likely due to the effect of the coating. Chitosan can reportedly form a tough, flexible coating layer with good tear resistance that exhibits improvement in all of the mechanical properties [36]. The chemical similarity of chitosan to the cellulose in Hanji is assumed to facilitate the formation of strong internal hydrogen bonds and produce a tough compatible fiber–binder system in the coated Hanji [22]. However, upon dilution, the fiber–binder system is weakened by the quantitative decrease in internal hydrogen bonds. Hence, the decreasing trend in the mechanical properties was observed with increasing dilution ratio and the resulting loss of already existing interfiber bonding networks in Hanji. 4.3.2. Oil resistance The Kit numbers of the samples, which represent the level of oil resistance, are shown in Figure 4. SC and SD showed the same Kit numbers as neat Hanji. However, 10
samples SA and SB showed increased Kit numbers; in particular, SA showed the highest Kit number. The obtained results are quite expected. From the data, it is possible to state that the CSNS coating improved the oil resistance of Hanji up to a dilution level of 1/10. The improved oil resistance of SA and SB compared to that of neat Hanji results from the CSNS coating, which forms a layer over the surface of Hanji and resists the passage of oil through it [35]. In addition, the CSNS that penetrated the internal voids of Hanji in the coating process would exhibit a positive effect by forming sufficient interfiber bonds with the cellulose in Hanji. These interfiber bonds can bridge the interfiber distances of the cellulose fiber surfaces and establish new bonds between the fibers and fibrils. Thus, a network-like system is created that would not have otherwise formed before CSNS coating; consequently, a physical barrier against oil penetration is developed and oil spread is arrested [22]. The lower oil resistance of SB compared to that of SA can be attributed to coating with the diluted CSNS, which quantitatively reduces the formation of additional new bonds and produces a weaker network than that in SA. Further, owing to the relatively high dilution, no further improvement in the oil barrier properties beyond neat Hanji is observed in SC and SD. 4.3.3. Antibacterial properties The inhibitory effect of neat Hanji and the coated Hanji samples (SA, SB, SC, and SD) is shown in Figure 5. As expected, no inhibition zone was observed for neat Hanji. However, SA and SB showed a clear inhibition zone of over 1 mm against E. coli. Compounds showing an inhibition zone of >1 mm are commonly considered good antibacterial agents according to the Swiss Norm (SN) 195920-ASTM E 2149-01[37]. Hence, the SA and SB samples obtained using the current approach can be considered good antibacterial agents. Further, to understand the positive influence of CSNS in imparting the antibacterial property to the Hanji, JIS Z 2801 standard was performed and the results are also shown in Figure 5. From the data, it is clear that irrespective of dilution all the coated samples showed higher antibacterial activity than neat Hanji (SA > SB > SC > SD> neat Hanji). This trend was expected, since it follows the trend of decreasing distribution of AgNPs with increasing dilution ratio, as confirmed by SEM and EDS. It is apparent that a stoichiometrically smaller population of AgNPs enables less significant interaction with bacterial colonies, resulting in decreased reduction in bacterial colonies with dilution. It is 11
also clear that, though neat Hanji and SD showed zero inhibition, yet the R (%) data, exhibited a clear difference, which showed 93.4 % higher antibacterial activity for SE than neat Hanji. This was evidently due to the effect of CSNS coating which could able to reduce bacterial growth effectively than neat Hanji. The antibacterial action is evidently due to CSNS coating, which is a combination of two antibacterial components, AgNPs and chitosan. The antibacterial activity of AgNPs is well known. Though the mechanism of the inhibitory effects of AgNPs on microorganisms is not completely clear, yet it could devastate causing leakage of lipopolysaccharide molecules and membrane proteins, leading to bacteria’s death [11]. The antibacterial action of chitosan is due to presence of charged groups in its backbone. These charged groups exhibit interactions with bacterial wall and results in the hydrolysis of peptidoglycans of microorganism wall and triggers the leakage of intracellular electrolytes, leading to the death of microorganism [38]. 5. Conclusion The functional properties of Hanji coated with a CSNS were investigated as a function of the dilution ratio. In the current approach, the CSNS was successfully prepared from AgNO3 and chitosan via ultrasonication and then applied to Hanji by a dip-coating method. UV-vis and TEM revealed the presence of AgNPs in the CSNS. FTIR spectroscopy indicated that the AgNPs were formed by the –OH groups of chitosan. SEM/EDS confirmed that the degree to which the CSNS coated the Hanji was in accordance with the dilution ratio. Improved tensile strength was observed for Hanji coated with the CSNS up to a dilution level of 1/10. In addition, improved burst strength was obtained by coating the CSNS up to a dilution level of 1/100. Further, it was found that a CSNS coating diluted by up to 1/10 of its original concentration can improve the oil resistance of Hanji. However, the Hanji exhibited improved antibacterial properties after it was coated with the CSNS even up to a dilution level of 1/1000. On the basis of the obtained results, it is possible to conclude that specific properties of Hanji can be improved by changing the dilution ratio. Further, among the four dilution ratios: undiluted, 1/10, 1/100 and 1/1000, ‘1/10’ can be considered as the ‘highest common dilution ratio’ that can improve all the four functional properties of Hanji. These significant findings have numerous versatile applications in Hanji-based packaging industries. 12
Acknowledgments This work was supported by the Korea Institute of Planning and Evaluation for Technology in the Ministry for Food, Agriculture, Forestry, and Fisheries of the Korean Government (No. IPET314050-3).
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Caption for Figures Scheme 1: Preparation of ultrasonicated chitosan–silver nanoparticle solution (CSNS) and its 17
application to Hanji Figure 1: a) UV-Vis spectrum of CSNS; b) TEM image of silver nanoparticles in CSNS; c) FTIR spectra of chitosan solution and CSNS Figure 2: SEM and EDS of neat Hanji, SA and SD Figure 3: Tensile and burst strength of neat Hanji, SA, SB, SC and SD Figure 4: Oil resistance of neat Hanji, SA, SB, SC and SD Figure 5: Antibacterial activity of neat Hanji, SA, SB, SC and SD
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