Preparation and Characterization of Cellulose Regenerated from

Nov 25, 2013 - Native cellulose has a highly crystalline structure stabilized by a strong intra- and intermolecular hydrogen-bond network. It is usual...
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Preparation and Characterization of Cellulose Regenerated from Phosphoric Acid Xuejuan Jia, Yingwen Chen, Chong Shi, Yangfan Ye, Peng Wang, Xiaoxiong Zeng, and Tao Wu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Nov 2013 Downloaded from http://pubs.acs.org on December 2, 2013

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Journal of Agricultural and Food Chemistry

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Preparation and Characterization of Cellulose

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Regenerated from Phosphoric Acid

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Xuejuan Jia, Yingwen Chen, Chong Shi, Yangfan Ye, Peng Wang, Xiaoxiong Zeng, Tao Wu*

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College of Food Science and Technology, Nanjing Agricultural University, Weigang 1, Nanjing

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210095, People’s Republic of China

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* Corresponding author. Tel./fax: +86 25 84396907. E-mail address: [email protected] (T.Wu).

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Abstract

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Native cellulose has a highly crystalline structure stabilized by a strong intra- and

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intermolecular hydrogen-bond network. It is usually not considered as a good gelling material

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and emulsion stabilizer due to its insolubility in water. Chemical modification is generally

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necessary to obtain cellulose derivatives for these applications. In this study, we have shown that

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by simply disruption the hydrogen-bond network of cellulose with phosphoric acid treatment, the

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regenerated cellulose can be a good gelling material and emulsion stabilizer. Microscopy, X-ray

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diffraction and Fourier transform infrared spectroscopy analysis have confirmed that the

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regenerated cellulose is primarily amorphous with low crystallinity in the structure of cellulose II.

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Stable aqueous suspensions and opaque gels that resist flowing can be obtained with the

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regenerated cellulose at concentrations higher than 0.6% and 1.6%, respectively. Moreover, it

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can effectively stabilize oil-in-water emulsions at concentrations less than 1% by a mechanism

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that combines network and Pickering stabilization.

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Keywords: Phosphoric Acid, Regenerated cellulose, Gel, Emulsion stabilization, Pickering,

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Network Stabilization

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Journal of Agricultural and Food Chemistry

Introduction

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Cellulose is the most abundant and renewable functional biopolymer created by nature; it

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exists in all higher plants, certain species of marine animals, bacteria, algae and fungi1. Native

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cellulose has a highly crystalline structure stabilized by a strong intra- and intermolecular

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hydrogen-bond network2. It is usually not considered as a good gelling material and emulsion

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stabilizer due to its insolubility in water3. Chemical modification is generally necessary to obtain

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cellulose derivatives with improved water solubility for applications of gelling and emulsion

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stabilization,

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hydroxypropylmethylcelluloses (HPMCs)3. Partial hydrolysis of purified wood pulp cellulose

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can obtain an insoluble cellulose material termed microcrystalline cellulose (MCC)4. Blending

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of MCC with anionic polysaccharides such as sodium CMC, xanthan or sodium alginate can

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obtain a colloidal form of MCC, which is water dispersible and can be used to stabilize foams

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and emulsions4.

such

as

carboxymethylcelluloses

(CMC),

methylcelluloses

(MC)

and

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Recently the gelling and emulsion stabilization properties have been explored in a new family

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of cellulosic materials - nanocelluloses5-8. The supramolecular structures of native cellulose have

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both amorphous and crystalline domains, in which the latter can be released by acid hydrolysis or

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mechanical treatments to produce various forms of nanocelluloses, such as nanocrystalline

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cellulose (NCC), microfibrillated cellulose (MFC), or nanofibrillated cellulose (NFC) depending

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on the preparation method and source7. By acid hydrolysis, the amorphous domain is hydrolyzed

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with hydrochloric acid or sulfuric acid, or, less frequently, with phosphoric acid, leaving the

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crystalline domain as NCC9. Due to the harsh reaction conditions used and the resulting acid

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degradation of the amorphous region, the yield of NCC is generally low and was reported to be

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approximately 30%, even after optimization of the reaction conditions10. By mechanical

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treatments, cellulose fibers are delaminated by high pressure homogenization, microfluidizers or

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sonication to produce MFC or NFC, usually after carboxymethylation or enzymatic pretreatment

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to decrease the adhesion between cellulose fibrils11. The energy consumption of the mechanical

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treatment is very high, reaching 25,000 kWh per ton in the preparation of MFC7.

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The morphology of the cellulose nanocrystals is generally needle or rod shaped with widths of

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a few nanometers and lengths varying from tens of nanometers to several micrometers2. They

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typically have the same crystalline structure as their original fibers, although transformation from

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cellulose I to cellulose II was occasionally found12. If prepared by HCl hydrolysis, the surfaces

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are weakly negatively charged, as a result of the carboxylic groups in the residual hemicelluloses

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that were not removed during purification13, 14. Due to the same reason, MFCs and NFCs are also

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weakly negatively charged. However, if prepared by H2SO4 hydrolysis, there is a stronger

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negative charge due to the introduction of sulfate ester groups during the preparation14. Because

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of these charges, cellulose nanocrystals present good colloidal stability and dispersibility. NCCs

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prepared with HCl can be dispersed by the thorough removal of acid from the hydrolysate14.

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NFCs prepared after a carboxymethylation pretreatment are readily dispersed by sonication at

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neutral pH and under salt-free conditions11. NCCs prepared with H2SO4 can be dispersed in

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aqueous solution with a pH ranging from 2 to 115. If prepared with H3PO4 hydrolysis, they are

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readily dispersible in polar solvents9. However, the colloidal stability of cellulose nanocrystals is

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sensitive to electrolytes due to specific interactions of counterions with the deprotonated

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carboxyl groups and the screening effect of the salt5. Nanocelluloses are excellent gelling

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materials and their gelling properties have been studied by several researchers15-17. Recently, the

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irreversible adsorption of cellulose nanocrystals at an oil-water interface to stabilize an emulsion

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by the “Pickering” effect was reported6,

8

and was explained based on the amphiphilic

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characteristic of cellulose nanocrystals residing in the crystalline organization at the elementary

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brick level6. These studies demonstrated the possibility of using nanocellulose for applications of

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gelling and emulsion stabilization.

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Compared with the extensive research on the crystalline form of cellulose, less research has

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been devoted to the amorphous form of cellulose. Although it is known that cellulose can be

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dissolved with solvents including SO2-diethylamine-dimethylsulfoxide (SO2-DEA-DMSO)

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solvent system18, N-methylmorpholine-N-oxide19, ionic liquid20 or alkali/urea aqueous systems21

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and subsequently regenerated with an anti-solvent, such as water, ethanol or acetone, to obtain a

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mixture of cellulose II and amorphous cellulose, its functional properties remain less thoroughly

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explored. In this paper, we used a benign solvent – phosphoric acid to dissolve cellulose and

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characterized the obtained functional material. We have shown by simply disruption of

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crystalline structure of cellulose with phosphoric acid, the regenerated cellulose is as versatile as

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the crystalline form of cellulose in gelling and emulsion stabilization.

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Experimental Details

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Materials. Dodecane (98%) was purchased from Aladdin (Shanghai, China) and purified by

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extensive extraction with water. Calcofluor White was purchased from Sigma-Aldrich

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(Shanghai, China). Original MCC powder (particle size of 20 - 100 µm), 85 wt% phosphoric acid

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(H3PO4) and all other chemicals were purchased from Sinopharm Chemical Reagent Co, Ltd.

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(Shanghai, China). Deionized water was used throughout the experiment.

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Dissolution and regeneration of MCC. Between 0.20 and 0.60 g original MCC powder was

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added to a 50 ml centrifuge tube, then 0.6 ml of deionized water was added to wet the cellulose

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powder, followed by the addition of 10 ml of pre-cooled or pre-heated H3PO4 in aliquots of 4, 4,

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and 2 ml. The cellulose suspensions were always mixed by a vortex before the addition of H3PO4

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in the next step. In this way, homogeneous cellulose suspensions were obtained and then

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incubated for durations of up to 5 hr. The %transmittances of the cellulose suspensions at 610

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nm were measured using a spectrophotometer and used as indexes to show the extent of cellulose

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dissolution (the higher the %transmittance, the better the dissolution). The effects of H3PO4

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concentration, ratio of cellulose/H3PO4, incubation temperature and time on the dissolution of

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cellulose were studied in this way. Analysis of the by-product formation was conducted by

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scanning the UV-Vis spectra of the incubated cellulose suspensions, directly or after dilution,

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with a spectrophotometer (LabTech Bluestar A, Beijing, China). Dilution was conducted with

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deionized water, followed by centrifugation to remove the white cloudy precipitate. A scale-up

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reaction condition of 5℃ and 24 hr with cellulose/water/85 wt% phosphoric acid in a ratio of

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1:3:50 (w/v/v) was selected to prepare the regenerated cellulose, followed by repeated

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centrifugation and dialysis to remove the phosphoric acid until a constant pH was obtained. The

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purity of the MCC and the yield of the regenerated cellulose were determined by the anthrone

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method22. One-half milliliter of dispersed cellulose was reacted with 4.5 ml of anthrone reagent

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at 95℃ for 10 min, followed by dilution with anthrone reagent to a proper concentration before

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reading the absorbance at 620 nm. Three replicates were run for each condition.

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Microscopy. Optical, fluorescence and polarized light micrographs of the original MCC and

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the regenerated cellulose suspensions were captured by an Eclipse microscope equipped with a

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digital camera (Nikon 80i, Japan). Calcofluor White was used as a fluorescence dye of cellulose

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at a concentration of 0.1% w/v. Scanning electron microscopy (SEM) of the original MCC

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powder and freeze-dried regenerated cellulose was performed by an electron microscope (S4800,

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Hitachi, Japan) at an accelerating voltage of 30 kV. The samples were gold coated in an ion

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sputter coater for 2 min before observation.

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XRD and FTIR Analysis. The crystal structures and chemical properties were studied by X-

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ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). X-ray diffraction

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(XRD) patterns of the lyophilized samples were collected using an X-ray diffractometer (D8

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Adrance, Bruker AXS, Germany) equipped with Cu-Kα radiation (λ=1.5418 Å) over the range

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of 2θ from 8 to 80°. The crystallinity index (CI, %) was determined by Eq. (1) 23, 24.

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CI (%) = 100 (I Max-I Am) / I Max

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where I

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amorphous cellulose peak at 2θ = 18°. FTIR spectra of the original MCC and the regenerated

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cellulose after lyophilization were obtained on a Nicolet 6700 FTIR spectrophotometer using the

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KBr disk method (sample/KBr ratio, 1/100). The FTIR spectra were recorded over the range of

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4,000 - 400 cm-1 with a resolution of 4 cm-1 and 32 total scans.

Max

is the maximum intensity of the principal peak, and I

(1) Am

is the intensity of the

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Determination of the phosphorous content and degree of substitution in regenerated

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cellulose. The content of phosphorus in the lyophilized sample was determined by a Perkin

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Elmer Optima 2100 DV Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES)

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after microwave digestion with a mixture of nitric acid and hydrogen peroxide. The substitution

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degree (DS) of phosphorus was calculated by Eq. (2),

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DS (%) = (m1/31) ×100% / (m2/162)

(2)

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where m1 and m2 represents the content of phosphorus and the mass of cellulose, respectively;

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31 and 162 are the atomic mass of the phosphorus and molecular mass of cellulose monomer

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units, respectively. Three replicates were run for each testing.

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Stability of regenerated cellulose suspension. The effects of ionic strength and pH on the

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stability of the regenerated cellulose suspensions were studied at temperatures of 25℃ and 50℃.

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The suspensions were diluted to 0.6% w/v, and the ionic strengths and pHs were adjusted with

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solid NaCl, 1 M HCl or 1 M NaOH, respectively.

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Emulsion preparation. The emulsions were prepared by a disperser (T18 digital ULTRA-

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TURRAX, IKA, Germany) at 10,000 rpm for 3 min using cellulose suspensions as the aqueous

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phase and purified dodecane as the oil phase in a volume ratio of 9:1. A series of concentrations

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were diluted from the stock cellulose suspensions at a concentration of 1.6% w/v. Emulsions

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prepared with the original MCC at the same concentrations were used for comparison.

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Results and Discussion

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Dissolution and regeneration of cellulose. Phosphoric acid was selected as the solvent to

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dissolve cellulose. The dissolution mechanism can be described by an esterification reaction:

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cellulose – OH + H3PO4 → cellulose –O– PO3H2+H2O25. When water was used to regenerate

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cellulose, the reverse reaction occurred: cellulose –O– PO3H2 +H2O → cellulose – OH +

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H3PO426. Direct dissolution of cellulose in 85% phosphoric acid was difficult because the

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exterior of the dry cellulose powder dissolved rapidly and formed a viscous layer through which

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the solvent penetrated very slowly. Pre-wetting of the cellulose powder was necessary to achieve

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a quick dissolution, as was reported elsewhere25. As shown in Figure 1a, after pre-wetting, 0.2 g

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of cellulose was readily dissolved in 10 ml 85 wt% (final concentration equals to 80.2 wt%) ice-

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cold phosphoric acid within 30 min. Smaller amounts of cellulose at concentrations of 0.5 - 1.0%

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w/v were dissolved in phosphoric acid at a reduced concentration of 77.8 wt%. With further

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reduction of the phosphoric acid concentration, the solubilization of cellulose was significantly

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inhibited, as evidenced by the decrease in %transmittance (T610 nm) to 0.77, 1.81 and 5.40 for

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2%, 1% and 0.5% w/v cellulose, respectively (Figure 1a). Zhang et al. showed that ice-cold

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phosphoric acid (≥83%) dissolved MCC completely, whereas 77 wt% phosphoric acid only

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caused swelling of the cellulose. In anhydrous phosphoric acid, a liquid crystalline solution

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containing 38% w/w cellulose could be easily prepared27. Thus, the dissolution of cellulose in

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phosphoric acid depends on the acid concentration and the ratio of cellulose/acid.

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The dissolution of cellulose also depended on the reaction temperature, as shown in Figure 1b.

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With the increase of temperature from 5℃ to 75℃, the %transmittance of the cellulose solution

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decreased at all cellulose/phosphoric acid ratios, revealing a trend of decreasing solubility with

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increasing temperature. The interaction between water and dry cellulose is strongly exothermic28,

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which may explain this observation. However, reasons such as changes in the structure of water

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with temperature29, the increased contribution of the negative entropy of mixing with increased

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temperature29, or the altered interaction between cellulose and solvent at low temperature similar

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to in the cellulose NaOH/urea system cannot be ignored30. Further study is needed to investigate

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the interaction between phosphoric acid and cellulose at low temperature. Nevertheless, the

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maximum amount of cellulose that can be solubilized by phosphoric acid was limited, even at the

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lowest temperature. When the concentration of cellulose was increased to 3% w/v, insoluble

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particles suspended in the solution could be observed with the naked eye and led to a decrease in

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%transmittance; even a prolonged solubilization time did not improve the extent of solubilization

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(inset of Figure 1b). In addition to influencing the dissolution process, the temperature had a

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profound effect on the formation of by-products. When the cellulose was heated in phosphoric

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acid at 75℃ for 30 min or at 50℃ for an extended time (≥12 hr), the solution turned light brown.

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The UV-Vis analysis of the diluted reaction mixture revealed the presence of a compound with a

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characteristic absorbance peak at 285 nm (Figure 1c). In the spectrum of the undiluted reaction

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mixture, a second compound, which was responsible for the light brown color, was identifiable

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with a characteristic absorbance peak at 450 nm (inset 1 of Figure 1c). The formation of by-

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products was intensified at prolonged heating times (inset 2 of Figure 1c). Currently, the

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chemical structures of these by-products remain unknown. They are likely associated with the

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dehydration products of cellulose, such as 5-hydroxymethyfurfural and its polymerized

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products31. Based on these experiments, a scaled-up reaction condition at 5℃ with

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cellulose/water/85% phosphoric acid in a 1:3:50 ratio was selected for preparing the regenerated

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cellulose. A heating time of 24 hr, instead of 30 min, was selected to ensure that all MCC

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particles were completely dissolved. Compared with the optimized yield (30%) of the cellulose

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nanocrystals prepared by hydrochloric acid or sulfuric acid hydrolysis10, the yield of our product

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was remarkably high, consistently reaching 86.6% (Table 1), suggesting that our phosphoric acid

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treatment is minimally destructive to the cellulose chain, and both the crystalline and amorphous

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domains are recovered. A recent publication showed that with phosphoric acid hydrolysis,

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thermally stable cellulose nanocrystals can be obtained at yields between 76 and 80%9. In

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contrast with this study, our method is based on the dissolution ability of phosphoric acid for

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cellulose at lower temperature and not the hydrolytic activity at higher temperature.

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Visual and microscopic analysis of regenerated cellulose. The 1.6% w/v original MCC

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suspension was not stable and sedimentation occurred within a few minutes (Figure 2a). At

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concentrations higher than 0.6% w/v, the regenerated cellulose formed a homogeneous

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suspension (Figure 2b), which was stable for at least three months. However, as we discuss later,

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this stability does not arise from the electrostatic repulsion that stabilizes cellulose nanocrystals

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whose surfaces are negatively charged. With a further increase in the concentration to 1.6% w/v,

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an optically opaque gel that resisted flow was obtained (Figure 2c). The optically opaque

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characteristic suggests that large particles or wide filaments that can scatter light are present in

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the gel. MFC and NFC form a transparent gel because their gel units are so thin that they do not

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scatter light significantly7, 11, whereas NCC prepared by sulfuric acid forms a perfectly uniform

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dispersion in water at low concentrations and self-organizes to a liquid crystal above a critical

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concentration2. Thus, the regenerated cellulose we prepared has a gel-like characteristic similar

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to that of MFC and NFC. The gel-like characteristic of the regenerated cellulose is also similar to

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that of partially oxidized dispersed cellulose nanofibers32 and networked cellulose prepared by

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the dissolution of MCC in 70% sulfuric acid and regeneration in ethanol33.

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The optical, fluorescence and polarized light micrographs of the original MCC and the

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regenerated cellulose suspensions were significantly different, as observed by the presence of

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large crystals only in the former (Figure 3). It is too early to conclude that cellulose loses its

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crystallinity during the dissolution and regeneration process because the regenerated cellulose

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crystals may be too small to be observed by a light microscope with a practical resolution

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limitation of 1 µm. At concentrations higher than 8%, the self-assembly of NCC to form a liquid

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crystal that displayed birefringence patterns was reported17. However, the regenerated cellulose

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could not be prepared at such a high concentration because of its high viscosity. The SEM

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analysis revealed the presence of individual large particles in the original MCC (Figure 4a) but

245

not in the regenerated cellulose, where chunky aggregates were observed (Figure 4b&c).

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A recent publication has shown that the lyophilization process can induce self-organization of

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cellulose particles into a lamellar structured foam composed of aligned membrane layers within

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the concentration range of 0.5 to 1.0 wt %34. Therefore, the chunky aggregates in Figure 4b and

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Figure 4c were probably also formed by the same lyophilization-induced-concentration effect

250

because we use a similar concentration in our lyophilization process. These chunky aggregates

251

were composed of curved, entangled and aggregated filaments (Figure 4d), which suggested that

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the morphology of regenerated cellulose is probably filament-like with a long aspect ratio. Thus,

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they probably have a high tendency to aggregate during drying.

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X-Ray and FTIR analysis. The X-ray diffractograms of the original MCC and the

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regenerated cellulose are shown in Figure 5a. Five peaks were observed for the original MCC at

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2θ = 14.8°, 16.5°, 20.5°, 22.7° and 34.6°, which correspond to the (110), (110), (012), (200),

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and (004) crystallographic planes of cellulose I, respectively12. Compared with the original MCC,

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less crystallinity was observed for the regenerated cellulose at 2θ = 12.3° and 20.3°, which

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correspond to the (110) and (110) crystallographic planes of cellulose II12. The CI of the

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regenerated cellulose was calculated to be 35%, which was significantly less than that of the

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original MCC CI value of 83% (Table 1). In contrast, the cellulose nanocrystals are usually of

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high crystallinity. Depending on the sources and preparation methods, the CI value can reach

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91% for CNC prepared from cotton fibers35, 78% from recycled pulp36, 81-85% for MCC37, and

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54-74% for CNC from banana fibers38. Although some previous studies showed that treatment of

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cellulose with concentrated phosphoric acid at 50℃ led to a mixture of amorphous cellulose,

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cellulose I and cellulose II39, 40, others reported that cellulose regenerated from NaOH/urea and

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ionic liquid were mainly amorphous with a minor component of cellulose II41, 42. It was likely

268

that all of the crystalline domains of the cellulose chains were disrupted during the dissolution,

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whereas some might be realigned and converted to cellulose II during the regeneration and

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possibly during the drying as well. A similar observation has been made for the dissolution of

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MCC in 70% sulfuric acid and regeneration in ethanol33.

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The FTIR spectra of MCC and regenerated cellulose are shown in Figure 5b. The broad bands

273

of stretching vibrations of the OH groups and -CH2- were observed from 4000 - 2995 cm-1 and at

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2900 cm-1, respectively. The C-O-C bending was from 1170 - 1082 cm-1, and the C-H stretching

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vibration was from 1060 – 1050 cm-1, all of which match the major functional groups in

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cellulose43, 44. The two spectra were similar, proving that the chemical structure of cellulose

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remained the same after regeneration. There was an O-H bending of absorbed water at

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approximately 1640 cm-1 in the spectra44, which indicated that the water was not completely

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removed from the cellulose due to the strong interaction of cellulose and water34, 45. Compared

280

with the spectrum of the original MCC, the peak at 1430 cm-1, which is attributed to the CH2

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scissoring motion, disappeared after regeneration, and the relative peak intensity at 895 cm-1,

282

which is attributed to the C-O-C asymmetric stretching, was increased43. The band at 1430 cm-1

283

is designated as the crystalline absorption band and is more pronounced in cellulose I; the band

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at 895 cm-1 is designated as the amorphous absorption band and is more pronounced in cellulose

285

II and amorphous cellulose46, 47. Thus, we can conclude that the cellulose I of the original MCC

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was converted to amorphous cellulose and cellulose II during regeneration.

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Combining the results of XRD, FTIR and light microscopy, we can draw a firm conclusion

288

that the cellulose lost its crystallinity during the dissolution. During regeneration and probably

289

during drying as well, amorphous cellulose was either partially crystallized into cellulose II,

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which is more stable than cellulose I48, or associated with each other through hydrophobic

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interactions, which are the driving forces in an amorphous system29. This process is depicted by

292

the scheme in Figure 6.

293

Stability of the regenerated cellulose suspension. The stability of the regenerated cellulose

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suspension was tested under different conditions by visual observation (Figure 7). With an

295

increase in the ionic strength from 0 to 2 M, an increase in the pH from 1 to 10, and an increase

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in the temperature from 25℃ to 50℃, the regenerated cellulose was well-dispersed, and no

297

precipitation was found over three months. NCCs prepared by sulfuric acid hydrolysis present

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good colloidal stability at low ionic strength (Na+, 1 mM) and in the pH range of 2 to 11 but

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begin to aggregate at high ionic strength (Na+, 10 mM) and a pH of 12, resulting from the

300

screening of electrostatic repulsion5, whereas NCCs prepared by HCl hydrolysis only exhibit

301

good colloidal stability at very low ionic strength14. Nanofibrillated cellulose (NFC) prepared by

302

enzymatic pretreatment and mechanical disintegration is not stable at decreasing pH or

303

increasing salt concentration11.It is then concluded that regenerated cellulose suspensions are not

304

stabilized by electrostatic repulsion as cellulose nanocrystals are because they are insensitive to

305

the change of electrolytes and pH.

306

Elementary analysis by ICP-OES also supported this conclusion, as shown by the minimal

307

substitution of phosphate groups on the regenerated cellulose (Table 1). One possible

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stabilization mechanism for the regenerated cellulose suspension is the repulsive hydration

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interaction, which arises whenever water molecules strongly bind to a surface containing

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hydrophilic groups, such as phosphate and sugar groups49. During the dissolution and

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regeneration, charged impurities, such as hemicellulose, were removed, which was reported in

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the regeneration process by the ionic liquid, 1-allyl-3-methilimidazolium chloride50. The

313

resulting regenerated cellulose is not charged but is probably highly hydrated, so it is not

314

sensitive to electrolytes and pH variation. The temperature usually has little influence on the

315

microstructure of nanocylinder dispersions, although a significant change of the microstructure

316

of the CNC dispersion was observed between 35℃ and 40℃17. Increasing the temperature

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usually leads to a weakening of the hydration interactions due to the dehydration of sugar groups.

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In our study, temperature barely affected the stability of the regenerated cellulose suspension. It

319

is possible that the temperatures we tested were not high enough to cause the destabilization.

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Stabilization of oil in water (o/w) emulsion by regenerated cellulose. The effects of

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different concentrations of regenerated cellulose on the stabilization of o/w emulsions stored at

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room temperature are illustrated in Figure 8. In emulsions prepared with the original MCC, both

323

significant oiling-off (formation of a layer of pure oil) and creaming was observed at all

324

concentrations (Figure 8a). In comparison, no oiling-off was observed for emulsions stabilized

325

with regenerated cellulose, even at the lowest concentrations. Oiling-off is usually caused by

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coalescence, in which two or more emulsion droplets merge together to form a single large

327

droplet3. Therefore, the regenerated cellulose is very effective in preventing coalescence. This

328

function may be explained by the amphiphilic characteristic of cellulose, a recent topic of much

329

debate29. Previously, cellulose was not considered to be a good stabilizer for emulsions because

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it is insoluble in water due to the strong intermolecular hydrogen bonds. A recent publication

331

showed that neutral cellulose nanocrystals can effectively stabilize water in oil (w/o) emulsions

332

by irreversibly absorbing at the interface due to the amphiphilic character resulting from the

333

crystalline organization6. Another recent publication has shown that ionic liquid regenerated

334

cellulose can stabilize both o/w and w/o emulsions51. An earlier report showed that in a

335

regenerated cellulose chain, the equatorial position of the glucopyranose ring where the hydroxyl

336

bonds are located is hydrophilic, whereas the axial direction where the C-H bonds of the

337

cellulose are located is hydrophobic52. Although an earlier publication suggested that the

338

amphiphilic property of cellulose is caused by the presence of a nitrogen-containing impurity53, it

339

was unlikely in our study because the original MCC and the amorphous cellulose were of high

340

purity (Table 1).

341

Nevertheless, our study clearly showed that regenerated cellulose was adsorbed at the

342

interface. This conclusion was drawn from two observations: (1) When low concentration

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343

regenerated cellulose was used to stabilize the emulsions, creaming was observed, but the bottom

344

serum phase was clear and free of cellulose (Figure 8b), indicating that oil can attract the

345

cellulose from the water. (2) Fluorescence micrographs demonstrated that regenerated cellulose

346

was adsorbed at the interface (Figure 9a-f). In the fluorescence micrograph, large emulsion

347

droplets with a strong fluorescence surface were observed, demonstrating the adsorption of

348

regenerated cellulose at the surface because the fluorescence dye - Calcofluor White only binds

349

to cellulose. It is arguable that the crystalline portion of the regenerated cellulose was adsorbed at

350

the interface because of the previous report. However, we believe that the amorphous portion

351

might be adsorbed at the interface for two reasons: (1) the same emulsion droplet observed in the

352

optical light micrograph (Figure 9g) completely disappeared in the polarized micrograph (Figure

353

9h), indicating that no crystalline cellulose was present in the emulsion system; (2) the

354

crystalline index of the regenerated cellulose was low and was likely caused by the drying

355

process. The real crystallinity in the wet-state was probably much lower. Nevertheless, the

356

adsorption of the regenerated cellulose at the interface was clear.

357

At the lowest cellulose concentration, the droplet diameter was approximately 61 µm, whereas

358

a stable droplet diameter of 20 µm was observed for all the other concentrations irrespective of

359

the amount of cellulose used (Figure 9a-f). The regenerated cellulose at concentration of 0.11%

360

was enough to cover the surface of the oil droplets to prevent coalescence. A similar observation

361

has been made by a previous report when using neutral cellulose nanocrystals to stabilize

362

emulsions8. However, this surface coverage can only prevent coalescence but not creaming. A

363

relatively high concentration of amorphous cellulose, e.g., 0.84%, was needed to prevent

364

creaming. These extra celluloses are used to thicken the aqueous phase and to prevent creaming.

365

Nevertheless, this concentration is much lower than the typical usage of common food

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polysaccharide-based emulsifiers3. Previously, neutral cellulose nanocrystals prepared by the

367

hydrochloric acid hydrolysis of bacterial cellulose was found to stabilize emulsions by the

368

Pickering effect8, whereas sulfated nanocrystals did not show interfacial properties6. Another

369

author concluded that MFC from mangosteen stabilizes emulsions by forming a solid-like

370

network in the continuous phase, i.e., network stabilization combined with adsorbing at the

371

oil/water interface, Pickering stabilization54. Our study also showed that the stabilization

372

mechanism of regenerated cellulose is a combination of network stabilization and Pickering

373

stabilization.

374

In summary, we described a facile method to produce functional cellulosic material by the

375

dissolution of MCC using cold phosphoric acid followed by regeneration in water. Although

376

phosphoric acid has been used for cellulose decrystallization to improve enzymatic

377

saccharification, it is less frequently used in the preparation of functional cellulose material.

378

Only a very recent publication employed the hydrolytic activity of phosphoric acid at higher

379

temperatures to prepare thermally stable cellulose nanocrystals. Our study used phosphoric acid,

380

but relies on its dissolution ability for cellulose at lower temperatures, which has clear benefits

381

over the use of ionic liquid or organic solvents in such a process in terms of availability, cost and

382

“greenness”. The regenerated cellulose has a morphology and gel characteristics similar to those

383

of MFC and NFC, but it is neutral and is most likely highly hydrated, which makes the resulting

384

suspensions more stable against changes in pH, ionic strength and temperature. Moreover, the

385

preparation of regenerated cellulose does not consume as much energy as the preparation of

386

MFC and NFC. Similar to neutral cellulose nanocrystals, the regenerated cellulose can

387

effectively stabilize oil in water emulsion by adsorption at the interface, which prevents the

388

coalescence and thickening of the aqueous phase, preventing creaming. However, the yield is

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much higher than that of cellulose nanocrystals whose preparation is based on acid hydrolysis.

390

Compared to the extensive research on crystalline cellulose, the amorphous form cellulose

391

deserves more research input in the future.

392

Acknowledgements

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393

This work is supported by a startup fund (804080) from Nanjing Agricultural University and

394

projects funded by the Priority Academic Program Development of Jiangsu Higher Education

395

Institutions (PAPD), the General Program of National Natural Science Foundation of China

396

(31271829), and the Natural Science Foundation of Jiangsu Province (BK2012770).

397

References

398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424

1. Lima, M. M. D.; Borsali, R., Rodlike cellulose microcrystals: structure, properties, and applications. Macromol. Rapid Commun. 2004, 25, 771-787. 2. Habibi, Y.; Lucia, L. A.; Rojas, O. J., Cellulose nanocrystals: chemistry, self-Assembly, and applications. Chem. Rev. 2010, 110, 3479-3500. 3. McClements, D. J., Food Emulsions: Principles, Practice, and Techniques. 2nd ed.; CRC Press: New York, 2005; pp 122-146. 4. Tuason, D. C.; Krawczyk, G. R.; Buliga, G., Microcrystalline Cellulose. In Food Stabilisers, Thickeners and Gelling Agents, Wiley-Blackwell: 2009; pp 218-236. 5. Zhong, L. X.; Fu, S. Y.; Peng, X. W.; Zhan, H. Y.; Sun, R. C., Colloidal stability of negatively charged cellulose nanocrystalline in aqueous systems. Carbohydr. Polym. 2012, 90, 644-649. 6. Kalashnikova, I.; Bizot, H.; Cathala, B.; Capron, I., Modulation of cellulose nanocrystals amphiphilic properties to stabilize oil/water interface. Biomacromolecules 2012, 13, 267-275. 7. Klemm, D.; Kramer, F.; Moritz, S.; Lindstrom, T.; Ankerfors, M.; Gray, D.; Dorris, A., Nanocelluloses: a new family of nature-based materials. Angew. Chem. Int. Ed. 2011, 50, 54385466. 8. Kalashnikova, I.; Bizot, H.; Cathala, B.; Capron, I., New pickering emulsions stabilized by bacterial cellulose nanocrystals. Langmuir 2011, 27, 7471-7479. 9. Espinosa, S. C.; Kuhnt, T.; Foster, E. J.; Weder, C., Isolation of thermally stable cellulose nanocrystals by phosphoric acid hydrolysis. Biomacromolecules 2013, 14, 1223-1230. 10. Bondeson, D.; Mathew, A.; Oksman, K., Optimization of the isolation of nanocrystals from microcrystalline cellulose by acid hydrolysis. Cellulose 2006, 13, 171-180. 11. Fall, A. B.; Lindstrom, S. B.; Sundman, O.; Odberg, L.; Wagberg, L., Colloidal stability of aqueous nanofibrillated cellulose dispersions. Langmuir 2011, 27, 11332-11338. 12. Sebe, G.; Ham-Pichavant, F.; Ibarboure, E.; Koffi, A. L. C.; Tingaut, P., Supramolecular structure characterization of cellulose II nanowhiskers produced by acid hydrolysis of cellulose I substrates. Biomacromolecules 2013, 13, 570-578.

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13. Fras, L.; Laine, J.; Stenius, P.; Stana-Kleinschek, K.; Ribitsch, V.; Dolecek, V., Determination of dissociable groups in natural and regenerated cellulose fibers by different titration methods. J. Appl. Polym. Sci. 2004, 92, 3186-3195. 14. Araki, J.; Wada, M.; Kuga, S.; Okano, T., Flow properties of microcrystalline cellulose suspension prepared by acid treatment of native cellulose. Colloids Surf., A 1998, 142, 75-82. 15. Ishii, D.; Saito, T.; Isogai, A., Viscoelastic evaluation of average length of cellulose nanofibers prepared by TEMPO-mediated oxidation. Biomacromolecules 2011, 12, 548-550. 16. Paakko, M.; Ankerfors, M.; Kosonen, H.; Nykanen, A.; Ahola, S.; Osterberg, M.; Ruokolainen, J.; Laine, J.; Larsson, P. T.; Ikkala, O.; Lindstrom, T., Enzymatic hydrolysis combined with mechanical shearing and high-pressure homogenization for nanoscale cellulose fibrils and strong gels. Biomacromolecules 2007, 8, 1934-1941. 17. Urena-Benavides, E. E.; Ao, G.; Davis, V. A.; Kitchens, C. L., Rheology and Phase Behavior of Lyotropic Cellulose Nanocrystal Suspensions. Macromolecules 2011, 44, 89908998. 18. Ciolacu, D.; Ciolacu, F.; Popa, V. I., Amorphous cellulose - structure and characterization. Cellul. Chem. Technol. 2011, 45, 13-21. 19. Fink, H. P.; Weigel, P.; Purz, H. J.; Ganster, J., Structure formation of regenerated cellulose materials from NMMO-solutions. Prog. Polym. Sci. 2001, 26, 1473-1524. 20. Han, J.; Zhou, C.; French, A. D.; Han, G.; Wu, Q., Characterization of cellulose II nanoparticles regenerated from 1-butyl-3-methylimidazolium chloride. Carbohydr. Polym. 2013, 94, 773-781. 21. Liu, S.; Zhang, L., Effects of polymer concentration and coagulation temperature on the properties of regenerated cellulose films prepared from LiOH/urea solution. Cellulose 2009, 16, 189-198. 22. Morris, D. L., Quantitative determination of carbohydrates with Dreywood's anthrone reagent. Science 1948, 107, 254-5. 23. Daud, W. R. W.; Kassim, M. H. M.; Seeni, A., cellulose phosphate from oil palm biomass as potential biomaterials. BioResources 2011, 6, 1719-1740. 24. Segal, L.; Greely, J. J.; Martin, J. A. E.; Comrad, C. M., An empirical method for estimating the degree of crystalline of native cellulose using the X-ray diffractometer. Text. Res. J. 1959, 29, 786-794. 25. Zhang, Y. H. P.; Cui, J. B.; Lynd, L. R.; Kuang, L. R., A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 2006, 7, 644-648. 26. Zhang, J.; Zhang, J.; Lin, L.; Chen, T.; Zhang, J.; Liu, S.; Li, Z.; Ouyang, P., Dissolution of microcrystalline cellulose in phosphoric acid-molecular changes and kinetics. Molecules 2009, 14, 5027-5041. 27. Boerstoel, H.; Maatman, H.; Westerink, J. B.; Koenders, B. M., Liquid crystalline solutions of cellulose in phosphoric acid. Polymer 2001, 42, 7371-7379. 28. Kocherbitov, V. U., S.; Kober, M.; Jarring, K.; Arnebrant, T., Hydration of microcrystalline cellulose and milled cellulose studied by sorption calorimetry. J. Phys. Chem. B 2008, 112, 3728-3734. 29. Glasser, W. G.; Atalla, R. H.; Blackwell, J.; Brown, R. M., Jr.; Burchard, W.; French, A. D.; Klemm, D. O.; Nishiyama, Y., About the structure of cellulose: debating the Lindman hypothesis. Cellulose 2012, 19, 589-598.

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30. Cai, J.; Zhang, L., Rapid dissolution of cellulose in LiOH/Urea and NaOH/Urea aqueous solutions. Macromol. Biosci. 2005, 5, 539-548. 31. Mascal, M.; Nikitin, E. B., Direct, high-yield conversion of cellulose into biofuel. Angew. Chem. Int. Ed. 2008, 47, 7924-7926. 32. Crawford, R. J.; Edler, K. J.; Lindhoud, S.; Scott, J. L.; Unali, G., Formation of shear thinning gels from partially oxidised cellulose nanofibrils. Green Chem. 2012, 14, 300-303. 33. Hashaikeh, R.; Abushammala, H., Acid mediated networked cellulose: preparation and characterization. Carbohydr. Polym. 2011, 83, 1088-1094. 34. Han, J.; Zhou, C.; Wu, Y.; Liu, F.; Wu, Q., Self-assembling behavior of cellulose nanoparticles during freeze-drying: effect of suspension concentration, particle Size, crystal structure, and surface charge. Biomacromolecules 2013, 14, 1529-40. 35. Martins, M. A.; Teixeira, E. M.; Correa, A. C.; Ferreira, M.; Mattoso, L. H. C., Extraction and characterization of cellulose whiskers from commercial cotton fibers. J. Mater. Sci. 2011, 46, 7858-7864. 36. Filson, P. B.; Dawson-Andoh, B. E., Sono-chemical preparation of cellulose nanocrystals from lignocellulose derived materials. Bioresour. Technol. 2009, 100, 2259-2264. 37. Herrera, M. A.; Mathew, A. P.; Oksman, K., Comparison of cellulose nanowhiskers extracted from industrial bio-residue and commercial microcrystalline cellulose. Mater. Lett. 2012, 71, 28-31. 38. Cherian, B. M.; Pothan, L. A.; Nguyen-Chung, T.; Mennig, G.; Kottaisamy, M.; Thomas, S., A novel method for the synthesis of cellulose nanofibril whiskers from banana fibers and characterization. J. Agric. Food Chem. 2008, 56, 5617-5627. 39. Kumar, V.; Kothari, S.; Banker, G. S., Effect of the agitation rate on the generation of low-crystallinity cellulose from phosphoric acid. J. Appl. Polym. Sci. 2001, 82, 2624-2628. 40. Zhang, J.; Zhang, B.; Zhang, J.; Lin, L.; Liu, S.; Ouyang, P., Effect of phosphoric acid pretreatment on enzymatic hydrolysis of microcrystalline cellulose. Biotechnol. Adv. 2010, 28, 613-619. 41. Li, R.; Zhang, L.; Xu, M., Novel regenerated cellulose films prepared by coagulating with water: Structure and properties. Carbohyd. Polym. 2012, 87, 95-100. 42. Lan, W.; Liu, C.-F.; Yue, F.-X.; Sun, R.-C.; Kennedy, J. F., Ultrasound-assisted dissolution of cellulose in ionic liquid. Carbohyd. Polym. 2011, 86, 672-677. 43. Oh, S. Y.; Yoo, D. I.; Shin, Y.; Seo, G., FTIR analysis of cellulose treated with sodium hydroxide and carbon dioxide. Carbohydr. Res. 2005, 340, 417-428. 44. Johar, N.; Ahmad, I.; Dufresne, A., Extraction, preparation and characterization of cellulose fibres and nanocrystals from rice husk. Ind. Crops Prod. 2012, 37, 93-99. 45. Moran, J. I.; Alvarez, V. A.; Cyras, V. P.; Vazquez, A., Extraction of cellulose and preparation of nanocellulose from sisal fibers. Cellulose 2008, 15, 149-159. 46. Adsul, M.; Soni, S. K.; Bhargava, S. K.; Bansal, V., Facile approach for the dispersion of regenerated cellulose in aqueous system in the form of nanoparticles. Biomacromolecules 2012, 13, 2890-5. 47. Kuo, C.-H.; Lee, C.-K., Enhancement of enzymatic saccharification of cellulose by cellulose dissolution pretreatments. Carbohydr. Polym. 2009, 77, 41-46. 48. Isogai, A.; Atalla, R. H., Amorphous celluloses stable in aqueous media. Regeneration from SO2-amine solvent systems. J. Polym. Sci., Part A: Polym. Chem. 1991, 29, 113-119. 49. Israelachvili, J. N., Intermolecular and surface forces. 2nd ed.; Academic Press: New York, 1991; pp 122-133.

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516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531

50. Casas, A.; Alonso, M. V.; Oliet, M.; Santos, T. M.; Rodriguez, F., Characterization of cellulose regenerated from solutions of pine and eucalyptus woods in 1-allyl-3methilimidazolium chloride. Carbohydr. Polym. 2013, 92, 1946-1952. 51. Rein, D. M.; Khalfin, R.; Cohen, Y., Cellulose as a novel amphiphilic coating for oil-inwater and water-in-oil dispersions. J. Colloid Interface Sci. 2012, 386, 456-463. 52. Yamane, C.; Aoyagi, T.; Ago, M.; Sato, K.; Okajima, K.; Takahashi, T., Two different surface properties of regenerated cellulose due to structural anisotropy. Polym. J. 2006, 38, 819826. 53. Ardizzone, S.; Dioguardi, F. S.; Quagliotto, P.; Vercelli, B.; Viscardi, G., Microcrystalline cellulose suspensions: effects on the surface tension at the air-water boundary. Colloids Surf., A 2001, 176, 239-244. 54. Winuprasith, T.; Suphantharika, M., Microfibrillated cellulose from mangosteen (Garcinia mangostana L.) rind: Preparation, characterization, and evaluation as an emulsion stabilizer. Food Hydrocolloids 2013, 32, 383-394.

532

Figure 1. Effects of reaction conditions on the dissolution of MCC: (a) effects of phosphoric

533

acid concentration; (b) effects of ratios of MCC/phosphoric acid, incubation temperature and

534

time (inset); (c) Uv-Vis spectra of reaction mixture at different incubation temperatures

535

(incubation time was 30 min and dilution factor was 4); inset 1 of c is the spectra of reaction

536

mixture by different dilution factors (incubation temperature was 75℃ and incubation time was

537

5 hr); Inset 2 is the spectra of reaction mixture after different incubation time (incubation

538

temperature was 75℃ and dilution factor was 10). Data are represented as mean ± standard

539

derivation (n=3).

540

Figure 2. Photographs of the 1.6% w/v original MCC suspension (a), 0.6% w/v regenerated

541

cellulose suspension (b), and 1.6% w/v (c) regenerated cellulose gel.

542

Figure 3. Optical (top), fluorescence (middle) and polarized images (bottom) of the original

543

MCC (left) and regenerated cellulose (right). Scale bars: 100 µm.

Figure Captions

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544

Figure 4. SEM images of original MCC (a – scale bar of 500 µm) and regenerated cellulose (b –

545

scale bar of 500 µm, c – scale bar of 100 µm and d - scale bar of 10 µm).

546

Figure 5. XRD (a) and FTIR spectra (b) of original MCC and regenerated cellulose.

547

Figure 6. Schematic representation of gel formation mechanism of regenerated cellulose.

548

Figure 7. The stability of 0.6% w/v regenerated cellulose suspension at different pHs, ionic

549

strengths, and temperatures (graph on top was at 25℃; bottom was at 50℃).

550

Figure 8. o/w emulsions stabilized by the original MCC (a) and the regenerated cellulose (b) at

551

different concentrations: from left to right, the concentrations are 0, 0.05, 0.11, 0.21, 0.42, 0.63,

552

0.84, and 1.06% w/v; the pictures were taken after 24 h of storage at room temperature.

553

Figure 9. Fluorescence (a - f), optical (g), and polarized light (h) micrographs of o/w emulsions

554

stabilized by regenerated cellulose at different concentrations (a - f: 0.05, 0.11, 0.21, 0.42, 0.63

555

and 1.06% w/v, g – h: 0.21% w/v).

556

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557

Tables

558

Table 1. The purity, yield, CI and DS values of the original MCC and regenerated cellulose. Data

559

are represented as mean ± standard derivation (n=3). sample

purity

original MCC

96.4 ± 1.3%

regenerated

99.0 ± 0.3%

yield 85.6 ± 1.1%

CI

DS

83 ± 3%

0

35 ± 2%

0.0001

cellulose

0.0001%

560 561

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±

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562

Page 24 of 33

Figure Graphic

1.0

80

60

40

20

60

Absorbance

0.8 60 40

0

0

50

100

150

200

250

300

Heating Time (min)

Inset 1

2 1 0

200

300

400

0.4

2.0

500

600

700

800

Wavelength (nm)

2.5

0.2

Inset 2

1 hr 2 hr 3 hr 4 hr 5 hr

1.5 1.0 0.5 0.0

20

-0.5 200

0.0

220

240

260

280

300

320

340

Wavelength (nm)

0

-0.2

0 80

563

0X 2X 10 X

3

0.6

20

40

4

5°C 25°C 50°C 75°C

80

Absorbance

80

%Transimittance (610 nm)

%Transmittance (610nm)

100

5°C 25°C 50°C 75°C

5

c

100

b

0.5 % w/v 1.0 % w/v 2.0 % w/v

Absorbance

100

a

%Transimittance (610 nm)

120

78

76

74

phosphoric acid concentration(wt%)

72

70

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Amount of cellulose per 10 ml phosphoric acid (g)

200

250

300

350

400

450

500

Wavelength (nm)

564

Figure 1. Effects of reaction conditions on the dissolution of MCC: (a) effects of phosphoric

565

acid concentration; (b) effects of ratios of MCC/phosphoric acid, incubation temperature and

566

time (inset); (c) Uv-Vis spectra of reaction mixture at different incubation temperatures

567

(incubation time was 30 min and dilution factor was 4); inset 1 of c is the spectra of reaction

568

mixture by different dilution factors (incubation temperature was 75℃ and incubation time was

569

5 hr); Inset 2 is the spectra of reaction mixture after different incubation time (incubation

570

temperature was 75℃ and dilution factor was 10). Data are represented as mean ± standard

571

derivation (n=3).

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Journal of Agricultural and Food Chemistry

572 573

Figure 2. Photographs of the 1.6% w/v original MCC suspension (a), 0.6% w/v regenerated

574

cellulose suspension (b), and 1.6% w/v (c) regenerated cellulose gel.

25 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

575 576

Figure 3. Optical (top), fluorescence (middle) and polarized images (bottom) of the original

577

MCC (left) and regenerated cellulose (right). Scale bars: 100 µm.

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Page 27 of 33

Journal of Agricultural and Food Chemistry

578 579

Figure 4. SEM images of original MCC (a – scale bar of 500 µm) and regenerated cellulose (b –

580

scale bar of 500 µm, c – scale bar of 100 µm and d - scale bar of 10 µm).

581

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Page 28 of 33

12000

a

Original MCC Regenerated cellulose

Intensity (a.u.)

10000

8000

6000

4000

2000

0 20

40

60

80

2θ (degree)

582 70

b

Original MCC Regenerated cellulose

60

%Transimttance

50 40 30 20 10 0

4000

583 584

3000

2000

1000

Wavenumbers (cm-1)

Figure 5. XRD (a) and FTIR spectra (b) of original MCC and regenerated cellulose.

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586 587

Figure 6. Schematic representation of gel formation mechanism of regenerated cellulose.

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588

589 590

Figure 7. The stability of 0.6% w/v regenerated cellulose suspension at different pHs, ionic

591

strengths, and temperatures (graph on top was at 25℃; bottom was at 50℃).

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592 593

Figure 8. o/w emulsions stabilized by the original MCC (a) and the regenerated cellulose (b) at

594

different concentrations: from left to right, the concentrations are 0, 0.05, 0.11, 0.21, 0.42, 0.63,

595

0.84, and 1.06% w/v; the pictures were taken after 24 h of storage at room temperature.

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596 597

Figure 9. Fluorescence (a - f), optical (g), and polarized light (h) micrographs of o/w emulsions

598

stabilized by regenerated cellulose at different concentrations (a - f: 0.05, 0.11, 0.21, 0.42, 0.63

599

and 1.06% w/v, g – h: 0.21% w/v).

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600

Journal of Agricultural and Food Chemistry

Graphic for Table of Contents

601

602

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