Effect of heat treatment on thermal resistance, transparency and antimicrobial activity of sonicated ginger cellulose film

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Contents lists available atScienceDirect

Carbohydrate Polymers

journal homepage:www.elsevier.com/locate/carbpol

E ﬀ ect of heat treatment on thermal resistance, transparency and antimicrobial activity of sonicated ginger cellulose ﬁ lm

Hairul Abral

^a,

*, Jeri Ariksa

^a

, Melbi Mahardika

^a

, Dian Handayani

^b

, Ibtisamatul Aminah

^b

, Neny Sandrawati

^b

, Eni Sugiarti

^c

, Ahmad Novi Muslimin

^c

, Santi Dewi Rosanti

^c

aDepartment of Mechanical Engineering, Andalas University, 25163, Padang, Sumatera Barat, Indonesia

bLaboratory of Sumatran Biota, Faculty of Pharmacy, Andalas University, 25163, Padang, Sumatera Barat, Indonesia

cLaboratory of High Resistant Materials, Research Center for Physics, Indonesian Institute of Sciences (LIPI) Serpong, Indonesia

A R T I C L E I N F O

Keywords:

Antimicrobial activity Cellulose nanoﬁber Relaxation Ultrasonication Residual strain

A B S T R A C T

Transparentfilm with high thermal resistance and antimicrobial properties has many applications in the food packaging industry particularly packaging for reheatable food. This work investigates the effects of heat treatment on the thermal resistance, stability of transparency and antimicrobial activity of transparent cellulosefilm.

Thefilm from ginger nanocellulosefibers was prepared with chemicals and ultrasonication. The driedfilm was heated at 150 °C for 30, 60, 90, or 120 min. The unheated and sonicatedfilm had the lowest crystallinity index and the lowest thermal properties. After heating, thefilm became brownish-yellow resulting from thermal oxidation. The reheatedfilm had higher thermal resistance than unheatedfilm. Heating led to further relaxation of cellulose network evidenced by shifting of the XRD peak positions toward lower values. The antimicrobial activity decreased due to heating. Average opacity value increases after short heating durations. It was relatively stable for further heating.

1. Introduction

Due to the serious ecological concerns around the use of synthetic polymers for packaging bio-based and biodegradable replacements have become a focus of recent research (Abral, Hartono et al., 2018;

Gonzalez-Gutierrez, Partal, Garcia-Morales, & Gallegos, 2010). Cellu- lose nanopaperfilm is one of the most promising of these because of its renewability, ease of manufacture, light weight, low cost, and transparency (Abral, Lawrensius, Handayani, & Sugiarti, 2018). Many works have focused on uses of cellulose nanopaper includingflexible thin-film supercapacitors (Gao et al., 2013), optically transparentfilms (Hsieh, Koga, Suganuma, & Nogi, 2017), transparent and conductive paper (Hu et al., 2013) andflexible organic light-emitting diode displays from bacterial cellulose (Ummartyotin, Juntaro, Sain, & Manuspiya, 2012).

Cellulose nanoﬁber has low thermal expansion (Nogi et al., 2013), high thermal resistance and stability (Yagyu, Saito, Isogai, Koga, & Nogi, 2015).

For applications in some food packaging, healthcare and hygiene products stability of transparency and antimicrobial activity on heating at high temperatures for a long duration can be important (Fernandes et al., 2014; Sung et al., 2013). A previous study has compared the transparency of cellulose nanopaper from Sitka spruce wood chips with

polyethylene terephthalate (PET)film and found the nanopaper to be more stable when these polymers were heated to 150 °C (Nogi et al., 2013). At this temperature, PET rapidly became opaque with increasing cloudiness. One promising type of low-cost eco-friendly fiber for transparent cellulosefilms can be obtained from ginger tubers (Abral, Ariksa, Mahardika, Handayani, Aminah, Sandrawati, Pratama et al., 2020). These are abundantly available in Indonesia which produces a large quantity of this plant (Rafi, Lim, Takeuchi, & Darusman, 2013).

Gingerﬁber from agro-waste has antimicrobial activity and potential for applications in food packaging and food safety (Abral, Ariksa, Mahardika, Handayani, Aminah, Sandrawati, Sapuan et al., 2020;

Duncan, 2011). Ginger nanoﬁber has previously been partially characterized (Abral, Ariksa, Mahardika, Handayani, Aminah, Sandrawati, Pratama et al., 2020;Raﬁet al., 2013;Thomas, Gopi, Jacob, Haponiuk,

& Peter, 2018). However, there were no works reported on the stability of antimicrobial activity and transparency of the ginger celluloseﬁlm on heating.

Ultrasonication has been found to be an effective method to prepare the cellulose transparentfilm (Chowdhury & Hamid, 2016;Mahardika, Abral, Kasim, Arief, & Asrofi, 2018;Wong, Kasapis, & Tan, 2009). Ul- trasonic treatment under a prolonged acid exposure caused a partial dissociation of cellulose hydrogen bond networks in the cellulose

https://doi.org/10.1016/j.carbpol.2020.116287

Received 19 January 2020; Received in revised form 10 April 2020; Accepted 10 April 2020

⁎Corresponding author.

E-mail address:[email protected](H. Abral).

Available online 21 April 2020

T

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nanocrystals (CNCs), ultimately resulting in the delamination and dis- order of the cellulose crystalline structure, thus leading to a decrease in the width and thickness of the CNCs (Guo, Guo, Wang, & Yin, 2016).

Intensive sonication has a major impact on the chain bonding within the cellulose structure and breaks the cellulose microﬁbrils along its (200) planes, yet the Iβcrystalline structure is still retained with re- duced crystallinity (Li & Renneckar, 2011). The average molecular weight of bacterial cellulose shows a drastic fall at the beginning of ultrasonication (about 64% reduction in theﬁrst 10 min) (Wong et al., 2009). Cellulose chain structures can undergo a compression after ultrasonication treatment resulting in a decrease in interlayer spacing of cellulose network (Bandyopadhyay, Selbo, Amidon, & Hawley, 2005).

Heat treatment of the compressed cellulose structures can change in their mechanical and physical properties (Bhuiyan, Hirai, & Sobue, 2001;Kolar, Strlič, & Marinček, 2002;Zhang et al., 2018). However, the effects of the heating durations at 150 °C on the thermal resistance of the ginger cellulosefilm after ultrasonication have not been fully understood. An in-depth analysis of the oxidation mechanism for this gingerfilm due to heating is still limited.

The aim of this work was to study the thermal properties of ginger nanocellulose, transparency, antimicrobial activities of a transparent film prepared from this plant when heated at 150 °C for various durations. Thefilm was characterized usingfield emission scanning electron microscopy (FESEM), optical microscopy (OM), transparency, Fourier- transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD).

Transparency, thermal resistance, and antimicrobial activity were also measured.

2. Materials and Methods 2.1. Materials

The raw materials for ginger nanofiber were the same as those described in a previous study (Abral, Ariksa, Mahardika, Handayani, Aminah, Sandrawati, Pratama et al., 2020). The nanofibers were iso- lated from ginger tubers (species:Zingiber officinale) from a local market in Padang, Indonesia. Distilled water, toluene (C7H8), ethanol (C2H6O), sodium hydroxide (NaOH), sodium chlorite (NaClO2), acetic acid gla- cial (CH3COOH), and hydrochloric acid (HCl) were also used. Distilled water, toluene (99%), and ethanol 96% (Andeska Brand) used were analytical grade and supplied by the Andeska Laboratory, Padang, In- donesia. Sodium hydroxide (Brataco Brand) was purchased from PT.

Brataco, Padang, Indonesia. Technical grade 80% Sodium chlorite was obtained from Sigma-Aldrich Pte. Ltd. Singapore. Acetic acid (density 1 g/cm³) and hydrochloric acid fuming 37% were supplied by Merck KGaA, Darmstadt, Germany.

2.2. Preparation of ginger nanocellulose and unheated and heated transparent nanopaper

Preparation of ginger nanocellulose and transparent nanopaper was the same as that described in previous work (Abral, Ariksa, Mahardika, Handayani, Aminah, Sandrawati, Pratama et al., 2020).

2.2.1. Preparation of ginger nanocellulose

The method for nanofiber isolation consisted of chemical treatment and ultrasonication. The raw gingerfiber was soaked in a mixture of toluene and ethanol in the ratio 2:1. Thefiber was then dewaxed using a hot plate stirrer (Daihan Scientific MSH-200) for 48 h, 500 rpm at 50 °C, then rinsed (filtered through a 200 T mesh cheesecloth 74μm) with distilled water until pH 7 using 250 mL Buchner Funnel, Glass Flask with Vacuum Pump (Shanghai Boerkang Vacuum Electron CO.,LTD.). The suspension was poured into a Teflon plate to be dried in a drying oven (Universal Oven Memmert UN-55) for 20 h at 50 °C. The driedfibers were soaked with a 5% NaOH, then dewaxed using a hot plate stirrer for 4 h, 500 rpm at 50 °C. The suspension was rinsed

(filtered through a 200 T mesh cheesecloth 74μm) using vacuum suctionfiltration device with distilled water until pH 7. The suspension was poured into a Teflon plate to be dried in a drying oven for 20 h at 50 °C. The driedfiber was crushed and mashed with a small electrical blender (Blender HR2115/01, Philips-Indonesia). These fibers were soaked with a mixture of NaClO2 and CH3COOH in the ratio 4:1.

Reduction of the lignin in thefibers was conducted in a hot plate stirrer at 60 °C and 500 rpm for 2 h. Thefibers were then rinsed using vacuum suctionfiltration device with distilled water until pH 7. The suspension was hydrolyzed with 5 M HCl at 50 °C for 12 h, washed using distilled water until pH 7 through Whatmanfilter paper number 42 using vacuum suction then rehydrolyzed. Then ultrasonication using an ultrasonic cell crusher (Ningbo Yinzhou SJIA Lab Equipment Co., Ltd.) at 600 W was conducted for 60 min, the temperature was maintained below 60 °C. The cellulosefibers after this process were found to be nano sized with average dimensions of 54.3 nm.

2.2.2. Preparation of unheated and heated transparent nanopaper 15 mL of the suspension was dried on a Teflon plate in a drying oven for 20 h at 50 °C. These driedfilms were then heated at 150 °C for 0, 30, 60, 90, or 120 min in the drying oven (Memmert UN-55). The 0 min unheatedfilm was used as a control. All studiedfilms were stored in a closed desiccator at 25 °C and relative humidity (RH) 50% before characterization.

2.3. Characterization

2.3.1. OM and FESEM morphology

The transparentﬁlm surface of 5 mm x 5 mm rectangular samples from each treatment was observed by an optical microscope (SZX10 stereo microscope, Olympus). The FESEM samples were placed on the FESEM sample stub. All samples were coated with carbon followed by gold for two minutes using an argon plasma metallizer (sputter coater K575X) (Edwards Limited, Crawley, United Kingdom) to reduce the electron charge. A FEI NOVA NanoSEM 230 machine (FEI, Brno- Černovice, Czech Republic) was used at 10 kV at 50,000x magniﬁcation to optimize observation of the surface morphology of the sample.

2.3.2. Film transparency

The transparency of ﬁlms was characterized using a spectrophotometer (Shimadzu UV 1800, Japan) based on ASTM D 1003-00 [Standard test method for haze and luminous transmittance of transparent plastics]. Equal weight rectangular samples 10 mm x 25 mm were prepared and placed in the spectrophotometer using a transmittance spectrum of 400 to 800 nm. Transparency was calculated from the area under the transmittance spectrum. The determination was repeated three times.

2.3.3. FTIR

Prior to FTIR characterization, all dried samples were stored in a closed chamber with RH 50% for 24 h. PerkinElmer Frontier equipment was used to scan theﬁlm from 4000–600 cm^-1at 4 cm⁻¹resolution.

FTIR spectra were repeated three times for each sample.

2.3.4. XRD testing

Dried samples were stored in a closed chamber with RH 50% and 75% for 24 h before XRD characterization. X-ray diﬀraction testing was carried out using PANalytical Xpert PRO at 25 °C, 40 kV and 30 mA. The samples were scanned from 2θ= 10° to 60°. The crystallinity index (Icr) percentage was measured using Eq. (1) (Segal, Creely, Martin, &

Conrad, 1958):

= −

I I I

I x

(%) ( )

cr 200 am 100

200 (1)

where I200 is the main peak at 2θ= 22.5° corresponding to (2 0 0) crystal plane, and Iam(2θ= 19°) is the intensity of the peak of the

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amorphous fraction.

2.3.5. Thermogravimetry analysis (TGA) and derivative (DTG)

A thermal analysis instrument DTG-60 from Shimadzu serial number C30565000570 (Kyoto, Japan) equipped with a TA-60WS thermal analyzer, FC-60Aﬂow controller and TA-60 software was used to characterize the TGA, and DTG of 7-10 mg samples. Each sample was put into the instrument which was set up with a nitrogenﬂow rate of 50 mL/min and the heating rate was 10 °C/min from 25-500 °C.

2.3.6. Antimicrobial activity

The methods for measuring the antimicrobial activity of ginger nanopaperﬁlm was similar to that published previously (Abral, Ariksa, Mahardika, Handayani, Aminah, Sandrawati, Pratama et al., 2020).

Activity was tested againstStaphylococcus aureus(SA),Bacillus subtilis (BC), Escherichia coli(EC),Pseudomonas aeruginosa (PA) andCandida albicans(CA) using an agar diﬀusion method (Bauer, Kirby, Sherris, &

Turck, 1966). 100μL of a microbial suspension was spread on the medium; nutrient agar medium for bacteria and sabouraud dextrose agar medium for fungi. The ginger nanopaperfilm from various treat- ments was cut into 6 mm diameter circular discs, and then was placed on a petri dish with this inoculated medium. The plates were cultured for 24 h at 30 °C. The antimicrobial efficacy of thefilms was evaluated using the McFarland scale. Chloramphenicol (30μg/mL) and nystatin (100 unit/disk) were used as positive controls for bacteria and fungi respectively. Three replicate tests were carried out under the same conditions for eachfilm and the diameter of the visible inhibitory region formed around thefilm discs measured.

2.3.7. Statistical analysis

IBM SPSS Statistics 25.0 (IBM Corporation, Chicago, USA) was used to analyze experimental data. One-way analysis of variance (ANOVA) and a p-test were used to identify the significance of any differences in opacity between treatment durations. Duncan multiple range tests were subsequently used using a 95% (p≤0.05) confidence level.

3. Results and Discussion

3.1. OM and FESEM morphology, transparency as a function of heating duration

The appearance of thefilm surface from OM observation without and with heating for 120 min can be seen inFig. 1A(a-b). The heated film (b) displays more air bubbles (white arrow) than nonheatedfilm (a).Fig. 1A(c-d) indicates the similarity of the appearance of the sonicated nanofibers before (c) and after 120 min heating (d).Fig. 1B shows the transparency of the cellulosefilm against the background of a red rose after various heating durations. Before heating, the nanopaperfilm was colourless and transparent (Fig. 1B(a)) with an average opacity value of 30.92 ± 0.41 AUnm (Table 1). Thisfilm had a transmittance value for visible light at 650 nm of 84.7 ± 0.2% (Fig. 1C). After 30 min heating, the average opacity increased to 32.69 ± 0.61 AUnm. Sur- prisingly, the transmittance value of this film remained almost un- changed after longer heating at (83.5 ± 0.4%). The increasing opacity corresponds to an increase in the light scattering in thefilm due to more air pockets (Abral, Ariksa, Mahardika, Handayani, Aminah, Sandrawati, Sapuan et al., 2020). This phenomenon of air trapped within the in- ternal layer structure of nanopaperfilm increasing light scattering has been described previously (Zhu et al., 2013). The 120 min heatedfilm (Fig. 1B(e)) contained more visible air bubbles than nontreatedfilm (Fig. 1B(a)). The micro-sized air bubbles such as those shown in Fig. 1A(b) and B(e) (marked with yellow arrow) probably appeared as a result of the agglomeration of nano-sized cavities (white arrow) between cellulose molecular chains. When the temperature increased, the vibration of molecular chains and expansion of the cellulose chains also increased allowing migration and agglomerations of the cavities which

continue to increase in volume after heating for longer duration at 150 °C.Fig. 1B(f) shows heating of 150 °C for 30 min caused obvious shrinkage of transparent polyethylene food packaging (yellow arrow), but not of the nanopaper (white arrow). This result indicates superior thermal stability of the cellulose nanopaper which is due to strong intra- and intermolecular hydrogen bonding between the cellulose molecules. A long heating duration led to a slight change in the color of thefilms as shown inFig. 1B(b-e). The discoloration increased with increasing heating duration. Thefilm which was heated for the longest duration (120 min) turned a visibly brownish-yellow color as a result of the strongest oxidation (Sandoval-Torres, Jomaa, Marc, & Puiggali, 2010). This thermal oxidation is because long heating duration results in an increase in oxygen concentration being in contact with cellulose (Rychlý et al., 2004). Atoms in molecules of the 0 minfilm had the lowest electronegativity as evidenced by the lowest wavenumber value of C-O stretching vibration at 1014 cm^-1(Fig. 2e) (Kagarise, 1955). This non-heated film also had the highest wavenumber value of C-H stretching at 2899 cm^-1(Fig. 2c). The high and low wavenumber values probably correspond to the presence of the residual tensile and com- pressive strain in the molecular structure which was thermo- dynamically unstable. Heating for 120 min led to an increase in electron transfer from carbon atoms in the cellulose molecules to oxygen atoms in the air. The oxygen atoms undergo reduction, gaining electrons, while the carbon atoms undergo oxidation, losing electrons. This result is in agreement with a shift of the wavenumber of C-O stretching toward higher value (1028 cm^-1) after 120 min heating. Loss of water from the cellulose structure may contribute to an increase of the residual tensile stress in the deformed cellulose networks which also ac- celerates oxygen diffusion and oxidation (Bertali, Scenini, & Burke, 2016).

3.2. FTIR spectra

Eﬀect of temperature on the properties of the functional groups of cellulose can be observed using FTIR spectroscopy (Syafri et al., 2018).

Fig. 2a displays the average values of three measurements on FTIR spectra for each sample. Shape, band, and intensity of the spectrum changed with heating duration. The shape of the peak became sharper after heating.Fig. 2b is the band at about 3325 cm^-1corresponding to O-H stretching vibration (Abral, Dalimunthe et al., 2018). The nonheatedfilm had the strongest intensity and the broadest peak shape of thefilms. This is due to the strongest disruption of crystal structure due to ultrasonication (Chen et al., 2011). Heating 30 min resulted in a decrease in intensity and sharper peaks. The sharpness of the band relates to an increase in intra- and intermolecular hydrogen bonds and chain reordering. This was supported by the ratio between the absorbance bands at 3324/1320 cm^-1(seeTable 1) (Oh et al., 2005). Wa- venumber value shifted slightly from 3327 cm^-1 to 3326 cm^-1corre- sponding to a decreased molecular vibration. Longer heating duration continues to weaken the intensity which can be related to releasing the strained lattice, thus changing the molecular vibration as shown by increase and decrease in wavenumber values (Fig. 2b).Fig. 2c displays C-H stretching vibration in range 3000-2500 cm^-1. The peak position of the 0 minfilm was the most left with a wavenumber of 2899 cm^-1, the intensity was the strongest and the shape the broadest. These functional groups were under nonhomogeneous strain condition and the crystals highly disoriented. Fig. 2d displays band peaks at the region of 1644–1648 cm^-1associated with O–H bending of the absorbed water (Thiripura Sundari & Ramesh, 2012). A shift to lower wavenumber value was observed on the heatedfilm compared to non-heated one due to the increasing intra- and interconnection among the microfibrils via hydrogen bonding after heat treating (Anitha, Brabu, John Thiruvadigal, Gopalakrishnan, & Natarajan, 2012;Abral, Satria et al., 2019;Guerrero, Kerry, & De La Caba, 2014). The higher intersections of O-H groups cause lower molecular vibration or a decreased wavenumber. Heating of the film weakened the intensity of vibrational

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spectra resulting from a decrease in the number of free hydroxyls. The C-O stretching band at about 1020 cm⁻¹ is believed to be from the backbone structure of cellulose (Wiley & Atalla, 1987). This band had the lowest wavenumber position (1014 cm^-1) and the strongest intensity. This is probably because the 0 minﬁlm had the strongest molecular deformation from chemical and mechanical treatment (Kačuráková, Smith, Gidley, & Wilson, 2002; Li & Renneckar, 2011;

Tanpichai et al., 2012). Ultrasonication can compress cellulose networks leading to a decrease in interlayer spacing and the length of the bonds as well as an increase in lattice strain and degree of disoriented crystal. Consequently, the molecular vibration of the C-O bonds became lower resulting in the lower electronegativity (Kagarise, 1955). Some of the potential energy from deformation will be stored in the deformed chains. The polarization of the molecules was changed due to the disruption of their structures (Garmire, Pandarese, & Townes, 1963). After heating duration for 30 min the band shifted from 1014 cm^-1 to

1023 cm^-1, its shape became sharper and the band intensity decreased (see inset). This is due to the release of the stored energy and network restructuring to gain stabilization. The sharper peak corresponds to more reordered crystal structures resulting in an increase in crystal size.

This phenomenon was observed inFig. 2f displaying weaker intensity related to a decrease in the disordered fraction as heating duration increased (El Oudiani, Msahli, & Sakli, 2017). This result is consistent with the oxidation reaction in the 120 minﬁlm (Fig. 1B_e) which de- monstrated an increased wavenumber of C-O stretching after heat treating (Coseri et al., 2015; Kim & Choi, 2014). Shifting the wavenumber toward higher value exhibits an expansion of the chain structures as evidenced by an increase ind-interplane spacing at 2θ= 22.5°

inTable 1.

Fig. 1.A) OM transparentfilms with 0 min (a) and 120 min (b) treatment on black surface of a Teflon plate, and FESEM morphology of nanopaperfilm with 0 min (c) and 120 min (d); B) the appearance of the transparentfilm before (a) and after heating of 150 °C for 30 (b), 60 (c), 90 (d), 120 min (e); the appearance of nanopaper film and synthetic polymer after 30 min heating (f); C) light transmittance as a function of wavelength at 150 °C for the various heating durations.

Table 1

The crystallinity index (Icr) fromFig. 3, the temperature of maximum decomposition (Tm) fromFig. 4, absorbance ratio 3324/1320 cm^-1fromFig. 2, and transmittance (T) of visible light at 650 nm of all samples fromFig. 1C.

Heating duration (min) Icr(%) d-spacing [Å] at 2θ= 22.5° Opacity (AUnm)* T at 650 nm (%) absorbance ratio 3324/1320 cm^-1 Tm(^oC)

0 51 3.92 30.92 ± 0.41^a 84.7 ± 0.2^b,c 0.73 ± 0.04^a 328

30 59 3.94 32.69 ± 0.61^b 83.5 ± 0.4^a 0.74 ± 0.09^a 333

60 55 3.99 33.37 ± 0.04^c,d 84.9 ± 0.2^c 0.80 ± 0.001^a 333

90 60 3.96 32.82 ± 0.04^b,c 84.7 ± 0.2^b,c 0.77 ± 0.03^a 334

120 66 4.00 33.67 ± 0.05^d 84.3 ± 0^b 0.78 ± 0.03^a 333

* The mean diﬀerence is signiﬁcant at p≤0.05.^{a, b, c, d}.

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3.3. XRD pattern

Fig. 3displays XRD patterns of samples as a function of heating duration. Two mains peaks at 2θ=15° and 22.6° represent crystal re- gions (Syafri, Kasim, Abral, & Asben, 2019). Height of these peaks is a measure of the crystallinity index (Icr) of the ﬁber. The nonheated sample (0 minﬁlm) shows the broadest peak and lowest intensity. This is a result of the strongest deformation of molecular structures causing

the loss in periodicity, as is characteristic of a disordered, less crystalline specimen (Cao & Tan, 2004). In this case, the ultrasonication while the disintegration of the weakest section like disordered cellulose chains via microjets and shock from the violent collapse of the bubbles disrupts the ordered arrangement of unit cell structures (Assender &

Windle, 1998;Bandyopadhyay et al., 2005). Cellulose chain structures can undergo compression and tension, had a highly disordered fraction, a decreased interlayer spacing and the short length of the bonds Fig. 2.FTIR spectra after various heating durations at 150 °C. (a) the full spectrum from 4000-250 cm^-1, (b-f) sections of the spectrum expanded to show changes in peaks related to speciﬁc functional groups.

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(Bandyopadhyay et al., 2005). This results in a broader intensity peak shifted to a higher 2θvalue in XRD pattern (Amanov, Umarov, &

Amanov, 2018;Biswas, Sinha, & Chakraborty, 2016;Epp, 2016). After the longer heating duration, the position of the crystalline peak shifts to a smaller angle from 2θ= 22.6° (0 minﬁlm) to 2θ= 22.5° (120 min ﬁlm). Also, the height of these peaks tends to increase (see inset) which indicates less imperfections in the crystal lattice at least regarding the (002) plane (Cao & Tan, 2004). A similar phenomenon also occurred in (110) crystalline planes at 2θ= 15.5°. Heating for 120 min expanded

thed-interplane distance from 3.9 to 4.0 Å and increased in Icrfrom 51 to 66% (Table 1). This result is because while theﬁlm is at a high temperature the higher activation energy of molecular chains allows them to restructure. Consequently, hydrogen bonding is formed between lone pairs of electrons from F, O, or N atoms and H (Boon, Lim, &

Gong, 2018). The cellulose chains tend to arrange so each long mole- cule is connected by hydrogen bonds to the neighbouring chains, forming a highly ordered crystalline form (Nakagaito, Nogi, & Yano, 2010). This result is consistent with the FTIR spectra (Fig. 2) showing the shallower and sharper peak of OH groups after longer heating.

Meanwhile, a shift toward lower Bragg angle corresponds to the com- pressive stress relaxation of the cellulose chain structure due to lattice deformation from ultrasonication treatment (Epp, 2016;Huang et al., 2017). This is in agreement with the FTIR pattern (Fig. 2) presenting a shift of wavenumber for C−O stretching from 1015 cm^-1(0 minﬁlm) to 1023 cm^-1after 30 min heating duration as a result of relaxation mechanism.

3.4. Thermal properties

Fig. 4displays the thermal properties of the samples before and after heat treatment. Initially, the weight of all samples in the temperature range 60-150 °C decreases slightly due to evaporation of absorbed water (Fig. 4a) (Abral, Basri et al., 2019; Voicu et al., 2016). The samples showed different weight losses due to the different amounts of water evaporated. The 0 minfilm shows the highest evaporation due to its highest hydrophilic nature. This can be a result of the highest number of the free hydroxyl groups as shown inFig. 2. The maximum evaporation rate of thisfilm was 0.15%/min (Fig. 4b). This rate was decreased after reheatingfilm. As the temperature increased further, there was a sudden second weight loss which can be attributed to the decomposition of cellulose in the range 260-360 °C (Abral, Satria et al., 2019; Araújo et al., 2018). At the temperature over 360 °C a third weight loss was observed due to the thermal-oxidative decomposition of the char (Asrofiet al., 2018;Zhao et al., 2013). Heat-treated nanopaper showed higher thermal resistance than nontreated. The temperature of maximum decomposition (Tm) at second weight loss increased after heat treatment (seeFig. 4b andTable 1). After heating for 30 min Tmwas shifted from 328 °C (0 minfilm) to 333 °C (30 minfilm), an increase of 2%. This increased Tmvalue is attributable to an increase of both the crystallinity index and the amount of interlinking hydrogen bonding between the polymer chains (Table 1). Consequently, much more energy is required to decompose the chain structure (Islam, Yasin,

& Rehman, 2014; Kim, Eom, & Wada, 2010). This phenomenon is consistent with XRD pattern (Fig. 3) with a sharper peak and FTIR spectra (Fig. 2) with C−O stretching shifting toward higher wave- numbers after heat treatment.

3.5. Antimicrobial activity

Fig. 5shows the antimicrobial activity inhibition zones which ap- pear as transparent areas (one marked with yellow arrow) for two bacteria after different heating durations. Table 2 displays the dia- meters of these zones against all microorganism tested to studied samples. In this study, the ginger nanopaperfilm was found to be effective against gram-positive bacteria, gram-negative bacteria, and fungi with inhibition zones ranging from 5.01 ± 0.3 to 10.1 ± 0.9 mm from the edge of the paper discs with no significant difference observed between microorganism type. The antimicrobial activity of ginger nanopaper might be due to the numerous active ingredients present in ginger including terpene and ginger oil. The major antimicrobial compounds in terpene are sesquiterpene hydrocarbons and the phenolic compounds gingerol and shogaol (Zick et al., 2008). Some studies have reported that gingerol and shogaol are the most active antibacterial and antifungal components in ginger rhizomes (Atai, Atapour, & Mohseni, 2009;Giriraju & Yunus, 2013). Heating for different durations resulted Fig. 3.XRD patterns for all tested samples before and after heating for the

various duration at 150 °C.

Fig. 4.TGA (a) and DTG (b) curves for all tested samples before and after heating at 150 °C for each duration.

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in diﬀerences in the clear zone diameter. Heat treatment caused a slight reduction of inhibition zone probably be due to damage of the bioactive compounds. Gingerol, in particular, is known to be thermally labile (Semwal, Semwal, Combrinck, & Viljoen, 2015).

4. Conclusion

Various durations of 150 °C heating to produce a relaxation of the structure of transparent cellulosefilm had an influence on its thermal resistance, transparency and antimicrobial activity. The preheatedfilm had a disrupted crystal structure resulting from the chemical and ultrasonication used in the nanofiber preparation and had the lowest crystallization index (51%) and thermal properties (Tm= 328 °C).

Heating the disrupted cellulose structures under atmospheric conditions allowed a relaxation of the chain networks and reformation of new inter- and intramolecular hydrogen bonds, consequently, the thermal resistance of the reheatedfilm increased. Longer heating durations increased in the amount of crystallization of thefilm. The initial 30 min heating led to an increase in Tmfor 2%,film opacity about 5.7% and a reduction of antimicrobial activity but further heating resulted in little change. The lower C-O stretching vibration (lower electronegativity) resulting from the deformed unit cell of cellulose increased oxygen diffusion and oxidation. These results offer deeper insights into the mechanisms of cellulose chain restructuring after long heating duration and their impact on the functional properties of ginger cellulosefilm.

Declaration of Competing Interest

We declare that there is no conﬂict of interest.

CRediT authorship contribution statement

Hairul Abral: Conceptualization, Methodology, Supervision, Funding acquisition, Formal analysis, Validation, Writing - original draft, Writing - review & editing. Jeri Ariksa: Investigation, Data curation. Melbi Mahardika: Investigation, Data curation, Formal analysis.Dian Handayani: Investigation, Data curation, Formal analysis, Writing - original draft.Ibtisamatul Aminah:Investigation, Data curation, Formal analysis, Writing - original draft.Neny Sandrawati:

Investigation, Data curation.Eni Sugiarti:Investigation, Data curation, Formal analysis.Ahmad Novi Muslimin:Investigation, Data curation.

Santi Dewi Rosanti:Investigation, Data curation.

Acknowledgements

Acknowledgement is addressed to Directorate General of Higher Education for supporting research funding with project name Fundamental Research, World Class Research, grant number T/19/

UN.16.17/PT.01.03/WCR-Material Maju/2020.

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Fig. 5.Diameter of inhibition zone (see more transparent area marked with yellow arrow) against (a)Pseudomonas aeruginosa(PA) and (b)Staphylococcus aureus (SA).

Table 2

Antimicrobial activity of the nanopaper before and after various heating times.

Heating duration (min) Diameter of inhibition zone (mm) against microorganisms

SA* BC* EC* PA* CA*

0 7.5±0.4 8.1 ± 1.2 7.5 ± 0.3 6.8 ± 0.9 10.1 ± 0.9

30 6.2 ± 0.1 5.1±0.2 5.2±0.2 5.2±0.1 5.2±0.1

60 6.0 ± 0.5 5.3±0.3 5.0±0.3 5.3±0.4 5.0±0.3

90 5.7±0.3 5.3±0.1 5.3±0.4 5.9±0.5 5.3±0.5

120 5.3±0.3 5.4±0.3 5.4±0.2 5.8±0.2 5.4±0.4

Positive Control 24.5 ± 0.5 25.6 ± 0.1 26.9 ± 0.9 24.5 ± 1.1 25.9 ± 0.5

* Staphylococcus aureus(SA),Bacillus subtilis(BC),Escherichia coli(EC),Pseudomonas aeruginosa(PA) andCandida albicans(CA).

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