Chitin Nanofibers as Reinforcing and Antimicrobial Agents in

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Chitin Nanofibers as Reinforcing and Antimicrobial Agents in Carboxymethyl Cellulose Films: Influence of Partial Deacetylation Mei-Chun Li, Qinglin Wu, Kunlin Song, H N Cheng, Shigehiko Suzuki, and Tingzhou Lei ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.6b00981 • Publication Date (Web): 06 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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ACS Sustainable Chemistry & Engineering

Chitin Nanofibers as Reinforcing and Antimicrobial Agents in Carboxymethyl Cellulose Films: Influence of Partial Deacetylation Mei-Chun Li1, Qinglin Wu1,* Kunlin Song1, H. N. Cheng2, Shigehiko Suzuki3, Tingzhou Lei 4,*

1

School of Renewable Natural Resources, Louisiana State University AgCenter, Baton Rouge,

Louisiana 70803, United States 2

Southern Regional Research Center, USDA Agricultural Research Service, New Orleans, LA

70124, USA 3

Department of Environment & Forest Resources Science Faculty of Agriculture, Shizuoka

University, Shizuoka 422-8529, Japan 4

Henan Key Laboratory of Biomass Energy, Zhengzhou, Henan 450008, China

Corresponding Authors: Qinglin Wu. E-mail: [email protected]

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ABSTRACT

The development of edible, environmentally friendly, mechanically strong and antimicrobial biopolymer films for active food packaging has gained considerable interest in recent years. The present work deals with the extraction and deacetylation of chitin nanofibers (ChNFs) from crab shells and their utilization as reinforcing and antimicrobial agents in carboxymethyl cellulose (CMC) films. ChNFs were successfully isolated from the speckled swimming crab shells for the first time through the multistep procedures involving deproteinization, demineralization, depigmentation and mechanical disintegration. Afterwards, the partially deacetylated ChNFs (dChNFs) were obtained through alkali treatment. It was found that the partial deacetylation led to the exposure of more amino groups on the surface of dChNFs and thus remarkably improved their dispersion state in an aqueous solution. The ChNF/CMC and dChNF/CMC films comprising up to 10 wt % nanofibers were prepared through the solution casting method, and their performance was evaluated and contrasted in terms of mechanical properties and antimicrobial activities. The results showed that the dChNF/CMC films exhibited superior mechanical and antimicrobial performance over ChNF/CMC films at any loadings, demonstrating the importance of ChNF surface chemistry in the development of high performance ChNF/CMC films for antimicrobial food packaging application.

KEYWORDS: Carboxymethyl cellulose, Chitin nanofibers, Deacetylation, Interfacial interaction, Mechanical properties, Antimicrobial activity. 2 ACS Paragon Plus Environment

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INTRODUCTION Since the mid-twentieth century, petroleum-based polymers have been widely applied in different fields due to their versatility, good processability and excellent physico-mechanical properties. However, after service life, it takes several hundreds of years to decompose them in a natural environment, causing serious environmental problems, e.g., water contamination, landfill waste, soil degradation and animal death. Recently, significant efforts have been made to reduce the consumption of petroleum-based polymers as well as to minimize human-beings’ influence on the environment. The use of environmentally friendly, renewable, biocompatible and biodegradable biopolymers (e.g., cellulose, chitin, lignin, starch, hemicellulose, pectin, alginate, xanthum, soy protein, and guar gum) to replace petroleum-based polymers becomes critical for better environmental preservation and sustainable development.1-4 Cellulose is well-known as the most abundant biopolymer on the earth. In order to expand its applicability, different cellulose derivatives, e.g., cellulose acetate, methyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose (CMC) are produced. CMC is synthesized through etherification of cellulose using monochloroacetic acid under alkaline condition. It is an anionic cellulose derivative with Ocarboxymethyl (-OCH2-COOH) replacing part of the hydroxyl (-OH) groups on the backbone. Because of its high viscosity, nontoxicity, biocompatibility and biodegradability, CMC is usually used as a viscosity modifier in food, personal care products, paints, adhesives, drilling fluids and other applications.5 In recent years, the development of edible, environmentally friendly, mechanically strong and antimicrobial CMC films for active food packaging has received considerable interest in both academic and industrial communities. However, poor mechanical properties of CMC make them unsuitable for most of practical use. Consequently, different reinforcing agents, e.g., cellulose 3 ACS Paragon Plus Environment


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nanocrystals,6,7 graphene oxide,8,9 nanoclay,10 and layered double hydroxide11 have been incorporated to improve the mechanical performance. Meanwhile, microbial contamination is another concern for the active food packaging application. Superior antimicrobial activities are highly desired to inhibit the growth of spoilage and pathogenic microorganisms in fresh food as well as to extend their shelf life. One of the most efficient methods to control the microbial growth is the incorporation of antimicrobial agent. Recently, various natural and synthetic antimicrobial agents, e.g., Zataria multiflora essential oil,12 clove oil,13 lysozyme and lactoferrin,14 hexamethylene diamine-co-diethylenetriamine,15 silver nanoparticles,16 and copper nanoparticles17 were reported to effectively enhance the antimicrobial activity of CMC films. It is worth noting that although metallic nanoparticles (e.g., silver and copper nanoparticles) exhibited outstanding antimicrobial activities against both the Gram-positive and Gram-negative bacteria, the use of them as antimicrobial agents in active food packaging should be paid special attention. A recent research demonstrated that silver nanoparticles were harmful to human lung fibroblast cells, which induced mitochondrial dysfunction, giving rise to DNA damage and chromosomal aberrations.18 Chitin, 𝛽-(1-4)-poly-N-acetyl-D-glucosamine, the second most abundant biopolymer in nature after cellulose, is widely available in the exoskeleton of insect, shrimp, crab, lobster and crawfish shells as well as the cell wall of yeast and fungi.19 About 1010- 1011 tons are produced per year; however, most of them were discarded as industrial and food waste without effective utilization.20 Recent research has shifted towards the high-value utilization and application of chitin in agriculture, industrial, biological and biomedical fields. Structurally, chitin is known to form microfibrillar arrangements, which are strongly wrapped by protein in the exoskeletons of crustacea. Within each chitin microfibril, there are dozens of nanofibers with diameter ranging 4 ACS Paragon Plus Environment

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from 2 to 5 nm and length of about 300 nm.25 In recent years, several methods, i.e., electrospinning,21 ultrasonication,22 wet grinding,20,23-25 high pressure homogenization,25,26 and self-assembly through precipitation/regeneration using suitable solvents27-29 have been employed to effectively prepare chitin nanofibers (ChNFs) from both commercial chitin and shell of crustacea. The resultant ChNFs were usually 10-100 nm in diameter and several micrometers in length, exhibiting large aspect ratio and specific surface area. Owing to the nanoscale dimension, large aspect ratio and surface area and high elastic modulus, ChNFs acted as an ideal reinforcing agent in various polymer matrices, e.g., poly(methyl methacrylate),25 natural rubber,30 polysilsesquioxane-urethaneacrylate copolymers,31 acrylic resin,32 poly(ethylene oxide)33 and polyvinyl alcohol.29,34,35 Therefore, it is of great practical interest to utilize ChNFs as a potential reinforcing agent in CMC film. On the other hand, due to the presence of chemically active hydroxyl and acetyl amine groups on the surface, its surface characteristics can be tailored through acetylation and deacetylation. For example, Ifuku et al.32 chemically modified ChNFs with acetic anhydride to have more hydrophobic acetyl groups on the surface. They found that the acetylation of ChNFs enhanced the compatibility with the hydrophobic acrylic resin, leading to the improvement in the moisture resistance. On the contrary, the deacetylation of ChNFs using concentrated alkaline solution removed the acetyl groups, leading to the exposure of more amino groups on the backbone.21 When the degree of acetylation was reduced to less than 50%, chitosan was produced. Chitosan is well-known as an effective antimicrobial agent against the Gram-negative bacteria.36 Although the exact antimicrobial mechanism of chitosan is still unclear, it is usually accepted that the amino groups on the surface of chitosan play a critical role. These results have inspired us to use the ChNFs as potential antimicrobial agent in CMC film by tailoring the amount of amino groups on the surface of ChNFs via appropriate deacetylation.



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Recently, Hatanaka et al.37 reported the effectiveness of ChNFs derived from commercial chitin powders in reinforcing CMC matrix through the construction of strong electrostatic interaction at the interfaces. However, they did not concern how the surface chemistry of ChNFs, i.e., deacetylation degree would affect the mechanical performance and antimicrobial activity of ChNF/CMC films. The main objectives of this study are 1) to directly extract ChNFs from the speckled swimming crab shells and 2) to investigate the influence of partial deacetylation on the mechanical and antimicrobial properties of ChNF/CMC films. Multistep procedures involving deproteinization, demineralization, depigmentation and mechanical disintegration were applied to isolate the ChNFs from the speckled swimming crab shell for the first time. Afterwards, the partial deacetylation of ChNFs was conducted to prepare deacetylated ChNFs (dChNFs) in a concentrated sodium hydroxide solution. The ChNFs and dChNFs obtained were then characterized using Fourier transform infrared spectroscopy (FTIR), solid-state

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magnetic resonance (NMR), transmission electron microscopy (TEM) and rheometry. Finally, the ChNF/CMC and dChNF/CMC films with a variable weight ratio of nanofibers (i.e., 1, 5 and 10 wt%) were obtained via solution casting. Their mechanical properties and antibacterial activities were evaluated and contrasted. The fracture surface morphology observation and theoretical Young’s modulus modelling were performed to interpret the distinctive reinforcing behaviors of ChNFs and dChNFs in CMC matrix as well. EXPERIMENTAL SECTION Materials. Fresh speckled swimming crabs, Arenaeus cribrarius, were collected from Florida, USA. The crabs were steamed and peeled to remove meat completely. The obtained shells were then washed with water for several times and dried in an oven. Carboxymethyl cellulose (CMC, PACTM-L, white or tan powder, bulk density 40-55 lb/ft3) was supplied by Halliburton Company 6 ACS Paragon Plus Environment

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(Houston, TX, USA). Hydrochloric acid (assay 36.5~38%) was purchased from VWR Company (West Chester, PA, USA). Sodium hydroxide pellets were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (assay 95%) was purchased from Carolina Biological Supply Company (Burlington, NC, USA). All chemicals were used without further purification.

Figure 1. Schematic illustration of the preparation of ChNFs and dChNFs from speckled swimming crab shells. Extraction and deacetylation of ChNFs from the speckled swimming crab shells. The extraction of ChNFs from the speckled swimming crab shells involved multistep procedures of deproteinization, demineralization, depigmentation and mechanical disintegration according to the literatures,20,38 as schematically illustrated in Figure 1. Prior to the chemical treatments, the crab shells were physically crushed into fine powders using a laboratory high-speed rotor mill (Columbia International Technical Equipment & Supplies LLC, Irmo, SC, USA). The resultant powders were then treated with NaOH, HCl and ethanol solutions to remove proteins, minerals 7 ACS Paragon Plus Environment


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and pigments, respectively. The yield of chitin was calculated as 12 wt%. Subsequently, ChNFs were prepared from the purified chitin through mechanical disintegration methods, including wet grinding and high pressure homogenization. Typically, the purified wet chitin samples were first diluted into 1 wt% suspension with deionized water under vigorous mechanical stirring. The resultant suspension was then manually poured into a grinder (MKCA6-2J, Masuko Corp., Japan) equipped with a pair of grinding stone (type: GA6-80; diameter: 150 mm) at room temperature. Grinding treatment was carried out with a clearance gauge of -0.5 (corresponding to a 50 μm shift) from the zero point at a rotation speed of 1500 rpm for 5 times. Afterward, the grinded suspension was passed through a high-pressure homogenizer (Microfluidizer M-110P, Microfluidics Corp., Newton, MA, U.S.A.) equipped with a pair of Z-shaped interaction chambers (one 87 μm diamond and one 200 μm ceramic). High pressure homogenization was performed at a flow rate of approximate 100 ml/min with an operating pressure of 138 MPa for 3 times. The temperature was controlled by adding ice into the cooling bath. The partial deacetylation of ChNFs was carried out in 33 wt% NaOH solution under vigorous mechanical stirring at 100 ℃ for 3 h. The ChNF and dChNF suspensions obtained were then poured into regenerated cellulose dialysis tube, and placed in a large tank with excess deionized water for several days until the pH value reached 6.5~7. Finally, the suspension was adjusted to 1 wt% and stored in a refrigerator for further use. Preparation of ChNF/CMC and dChNF/CMC films. A series of ChNF/CMC and dChNF/CMC films with different nanofiber concentrations (i.e., 0, 1, 5 and 10 wt%, based on CMC weight) were prepared using the solution casting method. For example, a fixed amount of 1 wt% ChNF or dChNF suspension was mixed with a desired amount of deionized water under vigorous stirring for 1 h to obtain the homogeneous dispersion state. Subsequently, CMC 8 ACS Paragon Plus Environment

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powders were added into the ChNF or dChNF suspension. The mixtures were vigorously stirred until the CMC was dissolved completely. The resultant mixtures were then poured into glass petri dishes and dried under ambient conditions for seven days. Finally, the films were carefully peeled off from the glass petri dishes with a knife and stored in zipper bags for further characterization. The obtained films were designated as ChNFx/CMC and dChNFx/CMC, where x is the mass percent of nanofibers relative to CMC. The resultant films were 80-100 𝜇m in thickness depending on the concentration of nanofibers. Characterization of ChNFs and dChNFs. The ChNF and dChNF suspensions were dried in an oven under vacuum at 60 ℃. The chemical structure of the solid films was studied using FTIR and solid-state

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C NMR. FTIR spectra were collected in the absorbance mode using a Bruker

FTIR analyzer (Tensor-27, Bruker Optics Inc., Billerica, MA), equipped with a Zn/Se attenuated total reflectance crystal accessory. The wavenumber ranged from 600 to 4000 cm − 1 with a resolution of 4.0 cm−1. Solid-state 13C NMR measurements were carried out on a Bruker Avance 400 WB instrument operating at 100.6 MHz at room temperature. The CP-MAS experiments were performed with a relaxation delay of 4.0 s and a contact time of 2.0 ms. Magic angle spinning (MAS) was achieved at a rate of 4 kHz. The chemical shift was in ppm related to an external sample of tetramethylsilane (TMS). The ChNF and dChNF suspensions were further diluted to 0.1 wt% with deionized water at pH value of approximately 6.5. Their surface charge, suspension stability and viscosity were evaluated. The zeta potential values were measured using a ZetaTrac analyzer (MicroTrac Inc., Largo, FL, U.S.A.). Five replicates were analyzed for each sample and an average value was reported. The suspension stability was carried out through a natural precipitation method over 24 h. Digital photos were taken using a digital camera at different intervals. The steady-state viscosity was examined using a stress-controlled rheometer 9 ACS Paragon Plus Environment


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(AR 2000, TA Instrument Inc., New Castle, DE, USA) with a cone-and-plate geometry (cone angle: 20; diameter: 40 mm; truncation: 56 μm) at 25 ℃. The shear rate was gradually increased from 1 to 1000 s-1. The morphology of ChNFs and dChNFs was observed using TEM (JEM 1400, JEOL) at an accelerating voltage of 120 kV. For the preparation of sample, 1 wt% ChNF or dChNF suspension was diluted to 0.02 wt% with deionized water. Glow discharge was employed to treat the copper grids (CF-400-CU, Electron Microscopy Sciences, Hatfield, PA, USA) prior to the dropping ChNF and dChNF suspensions onto the grids. The samples were stained using a 2 wt% uranyl acetate solution for 2 min to improve the contrast on the photomicrograph. Mechanical properties of ChNF/CMC and dChNF/CMC films. Tensile tests were conducted at a speed of 10 𝜇m/s on the AR2000 rheometer with a solid clamp in tension mode. Each sample was cut into dumbbell shape according to the ASTM D638-V, and its thickness was measured using a Mitutoyo digimatic indicator. Five replicates were performed and the reported values were the averages. Fracture surface morphology of ChNF/CMC and dChNF/CMC films. A field emission scanning electron microscopy (FE-SEM, a FEI QuantaTM 3D FEG dual beam SEM/FIB system, Hillsboro, OR) was employed to observe the fracture surface morphology of ChNF/CMC and dChNF/CMC films from tensile tests. The samples were mounted on an aluminum stub and sputter coated with a layer of gold before observation. Antimicrobial activities of ChNF/CMC and dChNF/CMC films. Antibacterial activities of ChNF/CMC and dChNF/CMC films were evaluated against both the Gram-negative bacterium Escherichia coli (E. coli) and the Gram-positive bacterium Staphylococcus aureus (S. aurues) using the Kirby–Bauer antibiotic testing method.39,40 Briefly, the bacteria were cultivated in 10


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mL sterilized tryptic soy broth and incubated in an incubator at 37 °C. The cell density was monitored by measuring the absorbance at 600 nm of culture medium using a spectrophotometer. When the cell density achieved approximately 107 CFU/mL, the culture medium was taken out from the incubator and carefully spread on the surface of solidified agar plate using a sterile cotton swap. Meanwhile, the ChNF/CMC and dChNF/CMC films were cut into disks with a diameter of 0.5 inch (12.7 mm) and sterilized by UV irradiation for 15 min. The disks were then placed on the inoculated agar plates and cultivated in the incubator at 37 °C for 24 h. Finally, the incubated agar plates were examined for a clear zone of inhibition (ZOI) around the specimens. The width of ZOI was determined using a digital caliper. The average value was reported based on three replicates. RESULTS AND DISCUSSION

Figure 2. Chemical structure of ChNFs and dChNFs: (a) FTIR and (b) solid-state

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spectra. Characterization of ChNFs and dChNFs. The successful extraction and partial deacetylation of ChNFs from the speckled swimming crab shells were confirmed from the FTIR spectra, as shown in Figure 2a. The crab shell powders exhibited a strong absorption peak at 1403 cm-1 11 ACS Paragon Plus Environment


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originated from protein and a relatively weak absorption peak at 1652 cm-1 corresponding to the amide I band of chitin.20 After multistep extraction, the resultant ChNFs showed very different FTIR spectrum with the crab shell powders. Particularly, the strong absorption peak at 1403 cm-1 derived from protein disappeared, and the characteristic absorption peaks of chitin, i.e., OH stretching band at 3435 cm-1, NH stretching bands at 3258 and 3100 cm-1, 3435 amide I bands at 1654 and 1620 cm-1, amide II band at 1552 cm-1, and amide III band at 1310 cm-1 appeared.21,41 These observations confirmed the successful production of ChNFs from the crab shell powder. After the deacetylation, the dChNFs showed same absorption peaks with ChNFs; whereas the absorption peaks of amide bands were found to be notably weakened compared with ChNFs. Several absorption band ratios, such as A1655/A3450, A1655/A2875, A1560/A3450 and A1560/A2875 were used to determine the degree of acetylation (DA) for chitin/chitosan.41,42 The calculated A1655/A3450, A1655/A2875, A1560/A3450 and A1560/A2875 values for the present ChNFs and dChNFs, were 1.84/1.70, 2.29/1.72, 2.36/2.21 and 2.94/2.12, respectively. The dChNFs had much lower absorption ratio values than ChNFs, suggesting the successful removal of acetyl group on the backbone of ChNFs. Furthermore, it was reported that for chitosan, a peak at around 1590 cm-1 corresponding to the amino group (-NH2) could be seen in the FTIR spectrum.43 The absence of amino group peak might be due to the fact that the DA of dChNFs was still too high. As a result, the amide I and II peaks at 1620 and 1552 cm-1 were so strong that the amino group peak at 1590 cm-1 was largely overlapped. These phenomena well confirmed the partial deacetylation, i.e., the amide groups were partially deacetylated into amino groups and the amide groups still predominated over the amino groups in the dChNFs backbone. Many techniques, including infrared spectroscopy, 1H-NMR, 13C-NMR, UV-vis and elemental analysis were employed to calculate the DA of chitin. Among these methods, the solid-state 13C 12 ACS Paragon Plus Environment

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NMR is considered the most convenient and reliable method. Therefore, solid-state

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spectra of ChNFs and dChNFs were recorded, as shown in Figure 2b. The ChNFs exhibited the representative resonance peaks of chitin at 173.7 (C=O), 104.3 (C1), 83.4 (C4), 75.9 (C5), 73.7 (C3), 61.2 (C2), 55.5 (C6) and 23.2 ppm (CH3). After the partial deacetylation, these peaks were slightly shifted to 173.6, 104.2, 83.1, 75.6, 73.9, 60.9, 55.4 and 23.0 ppm, respectively. The DA of chitin could be calculated by measuring the integral of the methyl group (-CH3) divided by the integral of all the carbon atoms in the backbone according to the following equation:44 𝐷𝐴 = 6 𝐼𝐶𝐻3 ⁄(𝐼𝐶1 + 𝐼𝐶2 + 𝐼𝐶3 + 𝐼𝐶4 + 𝐼𝐶5 + 𝐼𝐶6 )

(1)

With the above equation and Bruker NMR software, the DA of ChNFs and dChNFs was calculated as 0.80 and 0.69, respectively. About 14% acetyl groups were removed, i.e., 14% amino groups were exposed; after the partial deacetylation treatment. It is worth noting that the DA of ChNFs was relatively low as well, which was most likely due to the fact that the multistep extraction processes involving alkali treatment also eliminated acetyl groups.45 As known, the amino group in chitosan has a pKa value of 6.2~7.0, depending on the degree of deacetylation and the conditions of measurement.46 It was also reported that the pKa value of primary amines could be as high as 9.5.47,48 The present ChNF and dChNF suspensions had average zeta potential values of +15.59±1.13 and +31.00±4.81 mV, respectively; indicating the successful protonation of amino groups on the surface of both ChNFs and dChNFs. Because of the presence of more amino groups on the surface of dChNFs, dChNFs were protonated more intensively, resulting in higher positive zeta potential value. Recently, Pereira et al.48 reported the shift of isoelectric point with the change in the DA of chitin nanowhishers (CNWs). They found that the CNWs with DA of 81% had an isoelectric point of pH = 7.6, which increased to pH =



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8.8, when the DA decreased to 60%; i.e., the CNWs with a lower DA were more easily protonated. These findings were in good agreement with our results.

Figure 3. Appearance and stability of (a) ChNF and (b) dChNF suspension as a function of precipitation time; and (c) their steady-state viscosity plots as a function of shear rate. The protonation of amino group generated positive charge (NH3+) on the sample surface, which made chitosan soluble in an acidic solution. However, because the DA of ChNFs and dChNFs was still higher than 0.5, both of them were not soluble in an aqueous solution (Figures 3a and 3b). However, it seems that their dispersion state in an aqueous solution greatly varied depending on the DA. As shown in Figure 3a, the ChNFs strongly aggregated in the form of clusters, which could even be observed by visual inspection. After the partial deacetylation, these clusters were hardly seen due to the improved dispersion state of dChNFs (Figure 3b). Their suspension stability was further evaluated over 24 h. It was found that the ChNF suspension 14 ACS Paragon Plus Environment

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rapidly precipitated within 5 min and reached equilibrium after 3 h. Although the dChNF suspension also spontaneously sedimented, it exhibited superior stability over ChNF suspension. Noticeable flocculation was only observed after standing for 3 h. The improved dispersion state as well as suspension stability were ascribed to the exposure of more amino groups on the backbone of dChNFs, which were protonated such that the positively charged particles were more dispersible in water and repelled each other through electrostatic repulsion. For charged particles, the electrostatic repulsion between adjacent particles and the distortion of the electrical double layer surrounding them played critical roles in the steady-state suspension’s viscosity.49,50 In the case of chitin crystalline suspension, it has been demonstrated that the effect of distortion of the electrical double layer on the viscosity was very limited, whereas the electrostatic repulsion between neighboring positively charged chitin crystallites was critical.51 Therefore, if the stronger electrostatic repulsion forces existed on the dChNF suspension due to the exposure of more amino groups on the backbone, lower steady-state viscosity would be observed for the dChNF suspension compared with the ChNF suspension. Figure 3c shows the steady-state viscosity plots as a function of shear rate for 0.1 wt% ChNF and dChNF suspension. As expected, the dChNF suspension showed much lower viscosity than ChNF suspension within the whole range of shear rates studied. For example, at a shear rate of 1 s-1, a viscosity decrease of almost one order of magnitude was found upon the partial deacetylation. These observations confirmed the presence of more amino groups on the backbone of dChNFs and the improved dispersion state of dChNF suspension due to the enhanced electrostatic repulsion as well.



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Figure 4. TEM micrographs of (a) ChNFs and (b) dChNFs. Scale bar: 2 𝜇𝑚. The improved dispersion state of ChNFs through the partial deacetylation was further evidenced through TEM observation, as shown in Figure 4. Similar to the results observed in Figure 3a, the ChNFs remarkably aggregated to form large clusters (Figure 4a). In comparison with ChNFs, dChNFs exhibited a better dispersion state due to the improved electrostatic repulsion (Figure 4b). It is believed that such difference in dispersion state would cause distinctive reinforcing behaviors in the polymer matrix. On the other hand, it was found that the partial deacetylation had no noticeable influence on the dimensions of ChNFs. Both ChNFs and dChNFs had a wide distribution of dimensions with 10 ~ 80 nm in width and 500 nm ~ 10 𝜇m in length. Recently, Salaberria et al.52 prepared ChNFs from yellow lobster wastes using dynamic high pressure homogenization method up to 40 homogenizing passes. The resultant ChNFs were 80 to 100 nm in width and 5 to 10 um in length, which were much larger than the present ChNFs 16 ACS Paragon Plus Environment

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prepared through a combination of wet grinding and high pressure homogenization, demonstrating the high efficiency of wet grinding on the defibrillation of ChNFs. The average aspect ratio of ChNFs and dChNFs was estimated to be as high as 83.6, which was larger than most of chitin nanomaterials reported previously, e.g., chitin nanowhiskers prepared through HCl hydrolysis from commercial chitin (~16),30 chitin nanocrystals prepared through TEMPOmediated oxidation of 𝛼-chitin powders from crab shell (~42.5),53 and ChNFs prepared through dynamic high pressure homogenizing of 𝛼-chitin from yellow lobster (~60).54 Such large aspect ratio was beneficial for the formation of a strong percolation network, leading to superior reinforcement in the polymer matrix with the presence of a very small amount of nanofibers. Overall, based on the above observations, it is concluded that the ChNFs and dChNFs had similar geometric dimensions but different DA, which allowed us to accurately evaluate how the partial deacetylation would influence the mechanical and antimicrobial properties of ChNF/CMC films.

Figure 5. Typical stress-strain curves for ChNF/CMC and dChNF/CMC films with different nanofiber concentrations. 17 ACS Paragon Plus Environment


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Table 1. Mechanical and antimicrobial properties of ChNF/CMC and dChNF/CMC films with different nanofiber concentrations. 𝛿

𝜀

E

(MPa)

(%)

(MPa)

Halpin-Tsai

Ouali

E. coli

R. aureus

Neat CMC

43.6±0.9

46.5±3.7

432±15

-

-

0

0

ChNF1/CMC

44.2±3.0

44.5±4.1

438±26

-

-

458

443 -

-

0.2

0

1.8

0

0.6

0.4

2.8

1.0

Ec (MPa)

Width of ZOI (mm)

Films

dChNF1/CMC

60.3±2.6

29.6±2.3

683±22

ChNF5/CMC

47.5±1.7

32.2±8.3

508±37 560

dChNF5/CMC

65.3±5.4

25.0±6.8

753±48

ChNF10/CMC

49.9±2.1

25.1±4.8

546±21 676

dChNF10/CMC

61.3±5.1

15.3±3.9

770±14

550

719

Mechanical properties of ChNF/CMC and dChNF/CMC films. The tensile tests were conducted to examine the influence of partial deacetylation on the mechanical properties of ChNF/CMC films. Figure 5 shows the typical stress-strain curves of neat CMC, ChNF/CMC and dChNF/CMC films with 1, 5 and 10 wt% nanofibers. From the stress-strain curves, the mechanical parameters, i.e., tensile strength (𝛿), Young’s modulus (E) and elongation at break (𝜀) were derived and are summarized in Table 1. It was observed that both ChNFs and dChNFs exhibited effective reinforcement in CMC films. For ChNF/CMC films, both the tensile strength and Young’s modulus were gradually increased as the ChNF concentration increased. The highest tensile strength (49.86 MPa) and Young’s modulus (546 MPa) were achieved, when 10 wt% ChNFs were incorporated, which were 1.14 and 1.26 times of those of neat CMC film. However, the elongation at break gradually decreased from 46.49% to 25.11% with the addition 18 ACS Paragon Plus Environment

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of ChNFs from 0 to 10 wt%. This phenomenon was consistent with the general trend for fiber reinforced polymer composites that the improvement in the tensile strength and Young’s modulus of polymer composites with the addition of fibers was usually coupled with reduction in the elongation. Recently, various nanofillers, i.e., cellulose nanocrystals (CNC),6,7 graphene oxide (GO),8-9 montmorillonite (MMT),10 and layered double hydroxide (LDH)11 were used for CMC film reinforcement. The tensile strength and Young’s modulus of ChNF/CMC films were lower than those of CNC/CMC and GO/CMC nanocomposite films, but higher than those of MMT/CMC and LDH/CMC nanocomposite films. It seems that the reinforcing capacity of these nanofillers in CMC matrix was directly related to their Young’s modulus. In case of elongation, it was found that the ChNF/CMC films exhibited the highest elongation values. This might be ascribed to the high aspect ratio of ChNFs. Such long ChNFs dispersed in CMC matrix could create a strong percolation network and effectively bridge a craze at the fracture interface, avoiding the premature rupture.55 In comparison with ChNFs, the dChNFs exhibited superior reinforcing capacity in CMC matrix. Particularly, even at 1 wt% dChNF concentration, the tensile strength and Young’s modulus were significantly improved to 60.25 and 683 MPa, respectively; which were 1.38 and 1.58 times of the values for neat CMC films. An approximately 36% increase in the tensile strength and 56% increase in the Young’s modulus were achieved compared with ChNF1/CMC films. The optimal concentration of dChNFs appeared at 5 wt%, for which the tensile strength and Young’s modulus were 65.32 and 753 MPa, respectively. With a further increase in the concentration of dChNF to 10 wt%, the Young’s modulus was slightly increased to 770 MPa, whereas the tensile strength was reduced to 61.32 MPa. Two possible reasons were proposed for these phenomena. First, there is usually an optimum concentration for the tensile strength of 19 ACS Paragon Plus Environment


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polymer nanocomposites; beyond which the tensile strength begins declining with further increase in the nanofiller loading. Second, the uniform dispersion of dChNFs in CMC matrix became more and more difficult with continuous increase in the dChNF loading, leading to the reduction in the reinforcing efficiency. The above observations demonstrated that the optimum concentration for dChNFs in reinforcing CMC matrix was 5 wt%. To better understand the reinforcement of ChNF in CMC films, the Halpin-Tsai and the Quali models were employed to predict the Young’s modulus of ChNF/CMC films. The Halpin-Tsai model is a classical mean-field mechanical model for simulating the stiffness of short fiber reinforced composites. Considering the ChNF/CMC films as randomly aligned discontinuous fiber lamina, their Young’s modulus (𝐸𝑐 ) can be calculated from Equation:56

𝐸𝑐 = 𝐸𝑚 [

3 1 + 𝜂𝐿 𝜀𝜑𝑓 5 1 + 2𝜂𝑇 𝜑𝑓 + ] 8 1 − 𝜂𝐿 𝜑𝑓 8 1 − 𝜂𝑇 𝜑𝑓

(2)

where, 𝐸𝑓 (𝐸 ) − 1 𝑚 𝜂𝐿 = 𝐸𝑓 (𝐸 ) + 𝜀 𝑚

𝜂𝑇 =

𝐸𝑓 (𝐸 ) − 1 𝑚

𝐸𝑓 (𝐸 ) + 2 𝑚

(3)

(4)

where 𝐸𝑓 is the Young’s modulus of ChNFs, 𝐸𝑚 is the Young’s modulus of neat CMC film, 𝜑𝑓 is the volume fraction of ChNFs, and 𝜀 is a shape parameter dependent on the fiber dimensions


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and orientation. For high aspect ratio fiber, the 𝜀 is equal to (0.5𝐴)1.8 , as proposed by Van Es;57 where A is the aspect ratio of ChNFs (83.6 as determined from TEM observation). The Ouali model58 is an extension of the classical series-parallel Takayanagi model59 with the inclusion of percolation theory. For ChNF/CMC films, the Ouali model can predict their modulus (𝐸𝑐 ) by considering three phases: non-percolating ChNF phase, percolating ChNF network, and CMC matrix.55 This model can be written as the following equation:

𝐸𝑐 =

(1 − 2𝜓 + 𝜓𝜑𝑓 )𝐸𝑓 𝐸𝑚 + (1 − 𝜑𝑓 )𝜓𝐸𝑓2 (1 − 𝜑𝑓 )𝐸𝑓 + (𝜑𝑓 − 𝜓)𝐸𝑚

(5)

where 𝐸𝑓 is the Young’s modulus of ChNFs, 𝐸𝑚 is the Young’s modulus of neat CMC film, 𝜑𝑓 is the volume fraction of ChNFs, and 𝜓 corresponds to the volume fraction of the percolating ChNF network, which is given as the following equation: 𝜓 = 0;

𝜓 = 𝜑𝑓 (

𝜑𝑓 < 𝜑𝑐

𝜑𝑓 − 𝜑𝑐 0.4 ) ; 1 − 𝜑𝑓

(6)

𝜑𝑓 < 𝜑𝑐

(7)

where 𝜑𝑐 is the critical percolation volume fraction determined by 0.7/A, and A is the aspect ratio of ChNF (83.6 as determined from TEM observation). By substitution of 83.6 into the above equation, the 𝜑𝑐 for ChNFs was calculated as low as 0.84 vol%, corresponding to 0.75 wt% when using 1.43 and 1.59 g/cm3 as the density values of ChNFs and CMC, respectively. Apparently, the weight percentage of ChNFs in all the studied ChNF/CMC films exceeded 0.75 wt%, and thus there should be a strong ChNF percolating network in CMC matrix if ChNFs could be uniformly dispersed. The Young’s modulus of a single ChNF (𝐸𝑓 ) was rarely reported. However, it was reported that the Young’s modulus of ChNF film/membrane ranged from 1.8 to 21 ACS Paragon Plus Environment


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8.8 GPa, depending on the measurement method, relative humidity, DA, crystallinity as well as the origin of chitin.25,31,60,61 Based on the previously reported values, an average value of 5.3 GPa for ChNF was taken for theoretical modelling in this work. The predicted modulus values are shown in Table 1. In the case of ChNF1/CMC films, the predicted modulus values using both the Halpin-Tsai and the Ouali models were within the range of experimental result. However, compared with the Halpin-Tsai model, the Ouali model yielded a closer prediction by taking account of the percolation network. Indeed, because the concentration of ChNFs exceeded its percolation threshold (0.75 wt%), the interaction between neighboring ChNFs through the physical entanglement and chemical bond was significant and could not be ignored. As the ChNF concentration increased to 5 and 10 wt%, both the HalpinTsai model and the Ouali model overpredicted the modulus values. This discrepancy mainly resulted from the poor dispersion state of ChNF in CMC matrix (to be confirmed by FE-SEM observations). The basic assumption used for deriving most of the micromechanical models, including the Halpin-Tsai model and the Ouali model required the fillers to be uniformly and continuously distributed within the matrix.56,58 Interestingly, the dChNF/CMC films exhibited much higher modulus values than predicted values using both the Halpin-Tsai model and the Ouali model at any concentrations. This is presumably ascribed to the improved interfacial interaction between dChNFs and CMC matrix. It was reported that when the interfacial bonding between reinforcing agent and polymer matrix was strong enough, an immobilized polymer layer surrounding the reinforcing agent was created.62,63 This led to a significant increase in the effective volume fraction of rigid phase, especially for nanoscale reinforcing agent. Therefore, the volume used for theoretical modelling was lower than the effective volume, and the theoretical predicted values were lower than the experimental values. 22 ACS Paragon Plus Environment

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Figure 6. FE-SEM micrographs of fracture surface of (a, b) ChNF1/CMC; (c, d) dChNF1/CMC; (e, f) ChNF5/CMC; (g, h) dChNF5/CMC; (i, j) ChNF10/CMC; (k, l) dChNF10/CMC films. Scale bar: a, c, e, g, i and k - 30 μm; b, d, f, h, j and l – 5 μm. Morphology and reinforcing mechanism of ChNF/CMC and dChNF/CMC films. The mechanical experimental and modelling results demonstrated that the dChNF/CMC films exhibited superior mechanical performance over ChNF/CMC films. It is well known that the mechanical performance of polymer nanocomposite were directly associated with the dispersion state of reinforcing agents and interfacial adhesion between reinforcing agent and polymer matrix. Therefore, to interpret the observed distinctive reinforcing efficiency of ChNFs and dChNFs in CMC matrix, their fracture surface morphology was observed using FE-SEM, as shown in Figure 6. The neat CMC film showed very smooth fracture surface (Figure S1). The incorporation of ChNFs and dChNFs from 1 to 10 wt% generated very distinctive fracture morphology. For ChNF1/CMC film (Figures 6a and 6b), an inhomogeneous fracture surface, 23 ACS Paragon Plus Environment


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including smooth phase (corresponding to the unfilled matrix), and relatively rough phase (corresponding to the ChNF-filled matrix) was clearly observed. The formation of ChNF clusters and highly entangled network (Figures 3a and 4a) were responsible for the poor dispersion state. However, no micro-sized fibers could be seen on the fracture surface, suggesting that the ChNFs were still in nanoscale distributed in CMC matrix at this loading level. At a concentration of 5 wt%, the fracture surface became rougher with the presence of considerable amounts of voids and microfibers (Figures 6e and 6f). It is believed that at this loading level, the ChNFs aggregated in the form of micro-sized fibers, which were further pulled out under an external force, leaving the observed voids. With further increase in the concentration of ChNF to 10 wt%, more significant ChNF aggregations and larger holes were clearly shown on the fracture surface (Figures 6i, 6j and S2a). In stark contrast to the ChNF/CMC films, the dChNF/CMC films exhibited continuous and homogeneous fracture surface without the appearance of micro-sized fibers and voids at any concentrations (Figures 6c, 6d, 6g, 6h, 6k, and 6l). The absence of microsized fibers and voids on the fracture surface strongly demonstrated that the dChNFs were well individualized in nanoscale and evenly distributed in the CMC matrix. A more magnified FESEM micrograph of fracture surface of dChNF10/CMC film is shown in Figure S2b. It was still difficult to observe nano-scale dChNFs at the fracture surface. It seems that most of dChNFs were cracked at the joint of interface rather than being pulled out, suggesting the strong interfacial adhesion between dChNFs and CMC matrix. Furthermore, the surface morphology of neat CMC and dChNF10/CMC films was also observed, as shown in Figure S3. It confirmed that the nanoscale and highly individualized dChNFs were uniformly dispersed in the CMC matrix (Figure S3b).


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Figure 7. Schematic illustration of the dispersion state and interfacial bonding for ChNF/CMC and dChNF/CMC films. On the base of the above observations, distinctive reinforcing mechanisms for ChNF/CMC and dChNF/CMC films were reasonably proposed, as schematically illustrated in Figure 7. The reinforcement of ChNFs in CMC matrix could be well understood by taking account of the intrinsic characteristics of ChNFs (e.g., large aspect ratio, nanoscale dimension and high Young’s modulus) and the formation of interfacial interaction between ChNFs and CMC matrix (e.g., hydrogen bonding between amide groups of ChNFs and hydroxyl groups of CMC, and electrostatic attraction between the positively charged quaternary ammonium groups of dChNFs and the negatively charged carboxylate groups CMC),37,64,65 as illustrated in Figure 7. However, the poor dispersion state of ChNFs in CMC matrix (Figures 6b, 6f, 6j and 7a) greatly reduced their interfacial area with CMC matrix and hindered the formation of percolation network, leading to the relatively low reinforcing efficiency. The partial deacetylation of ChNFs not only 25 ACS Paragon Plus Environment


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promoted the formation of highly entangled percolation network due to the improved dispersion state in CMC matrix (Figures 6d, 6h, 6l and 7b), but also enhanced their interfacial adhesion with CMC matrix as indicated from the mechanical experimental/theoretical modelling results and the fracture surface morphology observations. Such enhanced interfacial adhesion most likely resulted from the construction of stronger electrostatic attraction at dChNF-CMC interfaces due to the exposure of more amino groups on the surface of dChNFs. Consequently, when an external force was exerted on the dChNF/CMC films, the enhanced percolation network and interfacial bonding facilitated a more effective stress transfer from the soft CMC matrix to the rigid dChNFs, leading to the superior mechanical performance over ChNF/CMC films.

Figure 8. Antimicrobial activities of neat CMC, ChNF/CMC and dChNF/CMC films against E. coli (up) and S. aureus (down). Antimicrobial properties of ChNF/CMC and dChNF/CMC films. In addition to mechanical performance, the antimicrobial activity is another important factor for active food packaging application. The antimicrobial activities of ChNF/CMC and dChNF/CMC films against both the Gram-negative E. coli and Gram-positive S. aurues were examined, as shown in Figure 8. The 26 ACS Paragon Plus Environment

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width values of ZOI are summarized in Table 1. In the case of neat CMC film, there was no clear ZOI presence around the specimens, indicating that the neat CMC film had no antibacterial activity against both E.coli and S. aureus. After the addition of ChNFs and dChNFs, the ZOI gradually appeared surrounding the specimens, depending on the concentration of ChNFs and dChNFs as well as the type of bacterium. For example, for ChNF/CMC films with 5 and 10 wt% ChNFs, their width of ZOI were 0.2/0 and 0.6/0.2 mm against E. coli and S. aureus, respectively; whereas for dChNF/CMC films with 5 and 10 wt% dChNFs, their width of ZOI were 1.8/0 and 2.8/1.0 mm against E. coli and S. aureus, respectively. At a same concentration, the dChNF/CMC films exhibited larger ZOI than ChNF/CMC films against both E. coli and S. aureus, suggesting the enhanced antimicrobial activities through the partial deacetylation of ChNFs. Furthermore, it was found that both ChNF/CMC and dChNF/CMC films had more effective antibacterial activity against E. coli over S. aureus. The antibacterial activity of chitosan against the Gram-negative bacterium has been extensively reported in recent years. Their potential antimicrobial mechanisms were proposed and could be summarized as below: (i) the presence of cationic groups on the surface of chitosan could interact with the anionic groups on the cell membrane of Gram-negative bacterium, causing the leakage of intracellular constitutes; (ii) chitosan with low molecular weight entered into the cell nucleus, hindering the synthesis of RNA and protein; and (iii) chitosan could act as a chelating agent, which bound metals to inhibit the microbial growth and toxin production.66-69 Similar to the above postulated antimicrobial actions of chitosan, the ChNFs and dChNFs effectively inhibited the growth of the Gram-negative bacterium E. coli as expected. However, for the antimicrobial activity against the Gram-positive bacterium S. aureus, it is believed that the contribution of the first action was very limited because both ChNFs/dChNFs and S. aureus are 27 ACS Paragon Plus Environment


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positively charged, and therefore the latter two mechanisms dominated. This caused the observed lesser antimicrobial activity against S. aureus than E. coli. Besides, the dChNF/CMC films had superior antibacterial activities against both E. coli and S. aureus over ChNF/CMC films as designed. This phenomenon was interpreted by the exposure of more amino groups on the surface through the partial acetylation, which were protonated and bound with the anionic groups on the cell membrane of E. coli more intensively, causing the significant leakage of intracellular constituents. CONCLUSIONS ChNFs were successfully extracted from the speckled swimming crab shells through the multistep procedures involving deproteinization, demineralization, depigmentation and mechanical defibrillation. The partial deacetylation of ChNFs was further conducted using an alkaline solution to expose more amino groups on the backbone. The resultant ChNFs and dChNFs were characterized using FTIR, solid-state 13C NMR, TEM and viscosity measurements. The results showed that the DA values of ChNFs and dChNFs were 0.8 and 0.69, respectively. Approximately 14% amino groups were exposed after the partial deacetylation treatment. Furthermore, the dChNFs exhibited better dispersion in an aqueous solution than ChNFs due to the enhanced electrostatic repulsion. Both ChNFs and dChNFs were then incorporated into CMC matrix using the solution casting method. The dChNF/CMC films had superior mechanical properties over ChNF/CMC films at any loadings. Mechanical theoretical modelling results and FE-SEM observations indicated that the enhanced interfacial bonding via the construction of stronger electrostatic attraction at dChNF-CMC interface, the formation of more rigid percolation network, and the improved dispersion state were responsible for the superior reinforcing efficiency of dChNFs over ChNFs. Moreover, the dChNF/CMC films exhibited 28 ACS Paragon Plus Environment

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stronger antimicrobial activities against both E. coli and S. aureus than ChNF/CMC films due to the exposure of more amino groups on the surface of dChNFs. It is expected that the present work will not only enrich the high-value utilization of crab shell wastes, but also provide us with an alternative way to prepare edible, sustainable, environmentally friendly, mechanically strong and antimicrobial biopolymer films for the active food packaging application. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Fracture surface morphology of neat CMC film (Figure S1); fracture surface morphology of ChNF10/CMC and dChNF10/CMC films (Figure S2); surface morphology of neat CMC and dChNF10/CMC films (Figure S3). AUTHOR INFORMATION Corresponding Authors * E-mails: [email protected] and [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.



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ACKNOWLEDGMENT This collaborative study was carried out with support from the Louisiana Board of Regents [LEQSF-EPS (2014)-OPT-IN-37, LEQSF(2015-17)-RD-B-01], and the USDA National Institute of Food and Agriculture McIntire Stennis project [1000017]. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

REFERENCES (1) Gandini, A. Polymers from Renewable Resources: A Challenge for the Future of Macromolecular Materials. Macromolecules 2008, 41, 9491–9504. (2) Yu, L.; Dean, K.; Li, L. Polymer blends and composites from renewable resources. Prog. Polym. Sci. 2006, 31, 576–602. (3) Thakur, V. K.; Thakur, M. K.; Raghavan, P.; Kessler, M. R. Progress in Green Polymer Composites from Lignin for Multifunctional Applications: A Review. ACS Sustainable Chem. Eng. 2014, 2, 1072–1092. (4) Thakur, V. K.; Thakur, M. K. Recent Advances in Graft Copolymerization and Applications of Chitosan: A Review. ACS Sustainable Chem. Eng. 2014, 2, 2637–2652. (5) Biswal, D. R.; Singh, R. P. Characterisation of carboxymethyl cellulose and polyacrylamide graft copolymer. Carbohydr. Polym. 2004, 57, 379–387. (6) Choi, Y.; Simonsen, J. Cellulose Nanocrystal-Filled Carboxymethyl Cellulose Nanocomposites. J. Nanosci. Nanotechnol. 2006, 6, 633–639. (7) El Miri, N.; Abdelouahdi, K.; Barakat, A.; Zahouily, M.; Fihri, A.; Solhy, A.; El Achaby, M. Bio-nanocomposite films reinforced with cellulose nanocrystals: Rheology of film-forming solutions, transparency, water vapor barrier and tensile properties of films. Carbohydr. Polym. 2015, 129, 156–167. (8) Yadav, M.; Rhee, K. Y.; Jung, I. H.; Park, S. J. Eco-friendly synthesis, characterization and properties of a sodium carboxymethyl cellulose/graphene oxide nanocomposite film. Cellulose 2013, 20, 687–698. (9) Son, Y.-R.; Rhee, K. Y.; Park, S.-J. Influence of reduced graphene oxide on mechanical behaviors of sodium carboxymethyl cellulose. Compos. Part B Eng. 2015, 83, 36–42. (10) Almasi, H.; Ghanbarzadeh, B.; Entezami, A. A. Physicochemical properties of starch– CMC–nanoclay biodegradable films. Int. J. Biol. Macromol. 2010, 46, 1–5. (11) Yadollahi, M.; Namazi, H.; Barkhordari, S. Preparation and properties of carboxymethyl cellulose/layered double hydroxide bionanocomposite films. Carbohydr. Polym. 2014, 108, 83– 90. 30 ACS Paragon Plus Environment

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(12) Dashipour, A.; Razavilar, V.; Hosseini, H.; Shojaee-Aliabadi, S.; German, J. B.; Ghanati, K.; Khakpour, M.; Khaksar, R. Antioxidant and antimicrobial carboxymethyl cellulose films containing Zataria multiflora essential oil. Int. J. Biol. Macromol. 2015, 72, 606–613. (13) Muppalla, S. R.; Kanatt, S. R.; Chawla, S. P.; Sharma, A. Carboxymethyl cellulose– polyvinyl alcohol films with clove oil for active packaging of ground chicken meat. Food Packag. Shelf Life 2014, 2, 51–58. (14) Barbiroli, A.; Bonomi, F.; Capretti, G.; Iametti, S.; Manzoni, M.; Piergiovanni, L.; Rollini, M. Antimicrobial activity of lysozyme and lactoferrin incorporated in cellulose-based food packaging. Food Control 2012, 26, 387–392. (15) Qian, L.; Guan, Y.; He, B.; Xiao, H. Synergy of wet strength and antimicrobial activity of cellulose paper induced by a novel polymer complex. Mater. Lett. 2008, 62, 3610–3612. (16) Siqueira, M. C; Coelho, G. F.; De-Moura, M. R.; Bresolin, J. D.; Hubinger, S. Z.; Marconcini, J. M.; Mattoso, L. H. C. Evaluation of antimicrobial activity of silver nanoparticles for carboxymethylcellulose film applications in food packaging. J. Nanosci. Nanotechnol. 2014, 14, 5512–5517. (17) Zhong, T.; Oporto, G. S.; Jaczynski, J.; Tesfai, A. T.; Armstrong, J. Antimicrobial properties of the hybrid copper nanoparticles-carboxymethyl cellulose. Wood Fiber Sci. 2013, 45, 215–222. (18) AshaRani, P. V; Low Kah Mun, G.; Hande, M. P.; Valiyaveettil, S. Cytotoxicity and Genotoxicity of Silver Nanoparticles in Human Cells. ACS Nano 2009, 3, 279–290. (19) Azuma, K.; Ifuku, S.; Osaki, T.; Okamoto, Y.; Minami, S. Preparation and biomedical applications of chitin and chitosan nanofibers. J. Biomed. Nanotech. 2014, 10, 2891–2920. (20) Ifuku, S.; Nogi, M.; Abe, K.; Yoshioka, M.; Morimoto, M.; Saimoto, H.; Yano, H. Preparation of Chitin Nanofibers with a Uniform Width as α-Chitin from Crab Shells. Biomacromolecules 2009, 10, 1584–1588. (21) Min, B.-M.; Lee, S. W.; Lim, J. N.; You, Y.; Lee, T. S.; Kang, P. H.; Park, W. H. Chitin and chitosan nanofibers: electrospinning of chitin and deacetylation of chitin nanofibers. Polymer 2004, 45, 7137–7142. (22) Fan, Y.; Saito, T.; Isogai, A. Preparation of Chitin Nanofibers from Squid Pen β-Chitin by Simple Mechanical Treatment under Acid Conditions. Biomacromolecules 2008, 9, 1919– 1923. (23) Ifuku, S.; Nogi, M.; Yoshioka, M.; Morimoto, M.; Yano, H.; Saimoto, H. Fibrillation of dried chitin into 10–20 nm nanofibers by a simple grinding method under acidic conditions. Carbohydr. Polym. 2010, 81, 134–139. (24) Ifuku, S.; Nogi, M.; Abe, K.; Yoshioka, M.; Morimoto, M.; Saimoto, H.; Yano, H. Simple preparation method of chitin nanofibers with a uniform width of 10–20 nm from prawn shell under neutral conditions. Carbohydr. Polym. 2011, 84, 762–764. (25) Chen, C.; Li, D.; Hu, Q.; Wang, R. Properties of polymethyl methacrylate-based nanocomposites: Reinforced with ultra-long chitin nanofiber extracted from crab shells. Mater. Design 2014, 56, 1049–1056. (26) Salaberria, A. M.; Fernandes, S. C. M.; Diaz, R. H.; Labidi, J. Processing of α-chitin nanofibers by dynamic high pressure homogenization: Characterization and antifungal activity against A. niger. Carbohydr. Polym. 2015, 116, 286–291. (27) Zhong, C.; Cooper, A.; Kapetanovic, A.; Fang, Z.; Zhang, M.; Rolandi, M. A facile bottom-up route to self-assembled biogenic chitin nanofibers. Soft Matter 2010, 6, 5298–5301.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 47

(28) Zhong, C.; Kapetanovic, A.; Deng, Y.; Rolandi, M. A Chitin Nanofiber Ink for Airbrushing, Replica Molding, and Microcontact Printing of Self-assembled Macro-, Micro-, and Nanostructures. Adv. Mater. 2011, 23, 4776–4781. (29) Kadokawa, J.; Takegawa, A.; Mine, S.; Prasad, K. Preparation of chitin nanowhiskers using an ionic liquid and their composite materials with poly(vinyl alcohol). Carbohydr. Polym. 2011, 84, 1408–1412. (30) Gopalan Nair, K.; Dufresne, A. Crab Shell Chitin Whisker Reinforced Natural Rubber Nanocomposites. 1. Processing and Swelling Behavior. Biomacromolecules 2003, 4, 657–665. (31) Ifuku, S.; Ikuta, A.; Hosomi, T.; Kanaya, S.; Shervani, Z.; Morimoto, M.; Saimoto, H. Preparation of polysilsesquioxane-urethaneacrylate copolymer film reinforced with chitin nanofibers. Carbohydr. Polym. 2012, 89, 865–869. (32) Ifuku, S.; Morooka, S.; Morimoto, M.; Saimoto, H. Acetylation of Chitin Nanofibers and their Transparent Nanocomposite Films. Biomacromolecules 2010, 11, 1326–1330. (33) Wu, J.; Lin, H.; Meredith, J. C. Poly(ethylene oxide) bionanocomposites reinforced with chitin nanofiber networks. Polymer. 2016, 84, 267–274. (34) Deng, Q.; Li, J.; Yang, J.; Li, D. Optical and flexible α-chitin nanofibers reinforced poly(vinyl alcohol) (PVA) composite film: Fabrication and property. Compos. Part A Appl. Sci. Manuf. 2014, 67, 55–60. (35) Nata, I. F.; Wu, T.-M.; Chen, J.-K.; Lee, C.-K. A chitin nanofibril reinforced multifunctional monolith poly(vinyl alcohol) cryogel. J. Mater. Chem. B 2014, 2, 4108–4113. (36) Rabea, E. I.; Badawy, M. E.-T.; Stevens, C. V; Smagghe, G.; Steurbaut, W. Chitosan as Antimicrobial Agent: Applications and Mode of Action. Biomacromolecules 2003, 4, 1457– 1465. (37) Hatanaka, D.; Yamamoto, K.; Kadokawa, J. Preparation of chitin nanofiber-reinforced carboxymethyl cellulose films. Int. J. Biol. Macromol. 2014, 69, 35–38. (38) No, H. K.; Meyers, S. P.; Lee, K. S. Isolation and characterization of chitin from crawfish shell waste. J. Agric. Food Chem. 1989, 37, 575–579. (39) Song, K.; Wu, Q.; Zhang, Z.; Ren, S.; Lei, T.; Negulescu, I. I.; Zhang, Q. Porous Carbon Nanofibers from Electrospun Biomass Tar/Polyacrylonitrile/Silver Hybrids as Antimicrobial Materials. ACS Appl. Mater. Interfaces 2015, 7, 15108–15116. (40) Zhou, S.; Wang, M.; Chen, X.; Xu, F. Facile Template Synthesis of Microfibrillated Cellulose/Polypyrrole/Silver Nanoparticles Hybrid Aerogels with Electrical Conductive and Pressure Responsive Properties. ACS Sustainable Chem. Eng. 2015, 3, 3346–3354. (41) Kasaai, M. R. A review of several reported procedures to determine the degree of Nacetylation for chitin and chitosan using infrared spectroscopy. Carbohydr. Polym. 2008, 71, 497–508. (42) Kumirska, J.; Czerwicka, M.; Kaczyński, Z.; Bychowska, A.; Brzozowski, K.; Thöming, J.; Stepnowski, P. Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan. Mar. Drugs 2010, 8, 1567–1636. (43) Paulino, A. T.; Simionato, J. I.; Garcia, J. C.; Nozaki, J. Characterization of chitosan and chitin produced from silkworm crysalides. Carbohydr. Polym. 2006, 64, 98–103. (44) Heux, L.; Brugnerotto, J.; Desbrières, J.; Versali, M.-F.; Rinaudo, M. Solid State NMR for Determination of Degree of Acetylation of Chitin and Chitosan. Biomacromolecules 2000, 1, 746–751. (45) MARCHESSAULT, R. H.; MOREHEAD, F. F.; WALTER, N. M. Liquid Crystal Systems from Fibrillar Polysaccharides. Nature 1959, 184, 632–633. 32 ACS Paragon Plus Environment

Page 33 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(46) Sorlier, P.; Denuzière, A.; Viton, C.; Domard, A. Relation between the Degree of Acetylation and the Electrostatic Properties of Chitin and Chitosan. Biomacromolecules 2001, 2, 765–772. (47) Elaissari, A. Colloidal polymers. Synthesis and characterization Marcel Dekker, New York, 2003. (48) Pereira, A. G. B.; Muniz, E. C.; Hsieh, Y.-L. Chitosan-sheath and chitin-core nanowhiskers. Carbohydr. Polym. 2014, 107, 158–166. (49) Smoluchowski, M. v. Theoretische Bemerkungen über die Viskosität der Kolloide. Kolloid-Zeitschrift 1916, 18, 190–195. (50) Watterson, I. G.; White, L. R. Primary Electroviscous Effect in Suspensions of Charged Spherical Particles. J. Chem. Soc., Faraday Trans. 2 1981, 77, 1115−1128. (51) Li, J.; Revol, J.-F.; Marchessault, R. H. Rheological Properties of Aqueous Suspensions of Chitin Crystallites. J. Colloid Interface Sci. 1996, 183, 365–373. (52) Salaberria, A. M.; Fernandes, S. C. M.; Diaz, R. H.; Labidi, J. Processing of α-chitin nanofibers by dynamic high pressure homogenization: Characterization and antifungal activity against A. niger. Carbohydr. Polym. 2015, 116, 286–291. (53) Fan, Y.; Saito, T.; Isogai, A. Chitin Nanocrystals Prepared by TEMPO-Mediated Oxidation of α-Chitin. Biomacromolecules 2008, 9, 192–198. (54) Salaberria, A. M.; Labidi, J.; Fernandes, S. C. M. Chitin nanocrystals and nanofibers as nano-sized fillers into thermoplastic starch-based biocomposites processed by melt-mixing. Chem. Eng. J. 2014, 256, 356–364. (55) Xu, X.; Liu, F.; Jiang, L.; Zhu, J. Y.; Haagenson, D.; Wiesenborn, D. P. Cellulose Nanocrystals vs. Cellulose Nanofibrils: A Comparative Study on Their Microstructures and Effects as Polymer Reinforcing Agents. ACS Appl. Mater. Interfaces 2013, 5, 2999–3009. (56) J. C. Halpin, J. L. Kardos, Polym. Eng. Sci. 1976, 16, 344-352. (57) Van Es, M. A.; Xiqiao, F.; van Turnhout, J.; van der Giessen, E. In Specialty Polymer Additives: Principles and Applications; Al-Malaika, S., Golovoy, A. W., Wilkie, C. A., Ed.; John Wiley & Son: Hoboken, NJ, 2001; pp 391−414. (58) Ouali, N.; Cavaille, J. Y.; Perez, J. Plast. Rubber Compos. Process. Appl. 1991, 16, 55−60. (59) Takayanagi, M.; Uemura, S.; Minami, S. J. Polym. Sci., Part C: Polym. Symp. 1964, 5, 113. (60) Mushi, N. E.; Butchosa, N.; Salajkova, M.; Zhou, Q.; Berglund, L. A. Nanostructured membranes based on native chitin nanofibers prepared by mild process. Carbohydr. Polym. 2014, 112, 255–263. (61) Ifuku, S.; Ikuta, A.; Egusa, M.; Kaminaka, H.; Izawa, H.; Morimoto, M.; Saimoto, H. Preparation of high-strength transparent chitosan film reinforced with surface-deacetylated chitin nanofibers. Carbohydr. Polym. 2013, 98, 1198–1202. (62) Dubief, D.; Samain, E.; Dufresne, A. Polysaccharide Microcrystals Reinforced Amorphous Poly(β-hydroxyoctanoate) Nanocomposite Materials. Macromolecules 1999, 32, 5765–5771. (63) Tang, L.; Weder, C. Cellulose Whisker/Epoxy Resin Nanocomposites. ACS Appl. Mater. Interfaces 2010, 2, 1073–1080. (64) Wang, J.; Somasundaran, P. Adsorption and conformation of carboxymethyl cellulose at solid–liquid interfaces using spectroscopic, AFM and allied techniques. J. Colloid Interface Sci. 2005, 291, 75–83. 33 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 47

(65) Zhang, L.; Jin, Y.; Liu, H.; Du, Y. Structure and control release of chitosan/carboxymethyl cellulose microcapsules. J. Appl. Polym. Sci. 2001, 82, 584–592. (66) Papineau, A. M.; Hoover, D. G.; Knorr, D.; Farkas, D. F. Antimicrobial effect of water‐ soluble chitosans with high hydrostatic pressure. Food Biotechnol. 1991, 5, 45–57. (67) Chien, R.-C.; Yen, M.-T.; Mau, J.-L. Antimicrobial and antitumor activities of chitosan from shiitake stipes, compared to commercial chitosan from crab shells. Carbohydr. Polym. 2016, 138, 259–264. (68) Fei Liu, X.; Lin Guan, Y.; Zhi Yang, D.; Li, Z.; De Yao, K. Antibacterial action of chitosan and carboxymethylated chitosan. J. Appl. Polym. Sci. 2001, 79, 1324–1335. (69) Cuero, R. G.; Osuji, G.; Washington, A. N-carboxymethylchitosan inhibition of aflatoxin production: Role of zinc. Biotechnol. Lett. 1991, 13, 441–444.


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For Table of Contents Use Only Chitin Nanofibers as Reinforcing and Antimicrobial Agents in Carboxymethyl Cellulose Films for Active Food Packaging: Influence of Partial Deacetylation Mei-Chun Li1, Qinglin Wu1,* Kunlin Song1, H. N. Cheng2, Shigehiko Suzuki3, Tingzhou Lei 4,*

Synopsis: We reported the development of edible, sustainable, mechanically strong and antimicrobial films for active food packaging using carboxymethyl cellulose and chitin nanofibers isolated from crab shells.



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TOC 55x47mm (300 x 300 DPI)

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Figure 1. Schematic illustration of the preparation of ChNFs and dChNFs from speckled swimming crab shells. 82x72mm (300 x 300 DPI)



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Figure 2. Chemical structure of ChNFs and dChNFs: (a) FTIR and (b) solid-state 13C NMR spectra. 135x71mm (300 x 300 DPI)


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Figure 3. Appearance and stability of (a) ChNF and (b) dChNF suspension as a function of precipitation time; and (c) their steady-state viscosity plots as a function of shear rate. 67x89mm (300 x 300 DPI)



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Figure 4. TEM micrographs of (a) ChNFs and (b) dChNFs. Scale bar: 2 µm. 67x101mm (300 x 300 DPI)


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Figure 5. Typical stress-strain curves for ChNF/CMC and dChNF/CMC films with different nanofiber concentrations. 67x55mm (300 x 300 DPI)



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Figure 6. FE-SEM micrographs of fracture surface of (a, b) ChNF1/CMC; (c, d) dChNF1/CMC; (e, f) ChNF5/CMC; (g, h) dChNF5/CMC; (i, j) ChNF10/CMC; (k, l) dChNF10/CMC films. Scale bar: a, c, e, g, i and k - 30 µm; b, d, f, h, j and l – 5 µm. 154x115mm (300 x 300 DPI)


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Figure 7. Schematic illustration of the dispersion state and interfacial bonding for ChNF/CMC and dChNF/CMC films. 135x148mm (300 x 300 DPI)



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Figure 8. Antimicrobial activities of neat CMC, ChNF/CMC and dChNF/CMC films against E. coli (up) and S. aureus (down). 135x58mm (300 x 300 DPI)


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Figure S1. FE-SEM micrographs of fracture surface of neat CMC film. Scale bar: (a) 30 µm and (b) 5 µm. 77x38mm (300 x 300 DPI)



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Figure S2. FE-SEM micrographs of fracture surface of (a) ChNF10/CMC and (b) dChNF10/CMC films. Scale bar: 2 µm. 77x38mm (300 x 300 DPI)


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Figure S3. FE-SEM micrographs of surface of (a) neat CMC and (b) dChNF10/CMC films. Scale bar: (a) 10 µm and (b) 5 µm. 77x38mm (300 x 300 DPI)


Chitin Nanofibers as Reinforcing and Antimicrobial Agents in

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