Cellulose-based composite macrogels from cellulose fiber and

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Cellulose-based composite macrogels from cellulose fiber and cellulose nanofiber as intestine delivery vehicles for probiotics Qian Luan, Weijie Zhou, Hao Zhang, Yuping Bao, Ming-Ming Zheng, Jie Shi, Hu Tang, and Fenghong Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04754 • Publication Date (Web): 09 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Journal of Agricultural and Food Chemistry

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Cellulose-based composite macrogels from cellulose fiber and

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cellulose nanofiber as intestine delivery vehicles for probiotics

3 4

Qian Luan1, Weijie Zhou2, Hao Zhang1, Yuping Bao1, Mingming Zheng1, Jie Shi1

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Hu Tang*,1, Fenghong Huang*,1

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1 Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Oil

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crops and Lipids Process Technology National & Local Joint Engineering Laboratory,

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Key Laboratory of Oilseeds processing, Ministry of Agriculture, Hubei Key

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Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, China

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2 Department of Chemistry, Stony Brook University, Stony Brook, NY11794

12

*

Corresponding author: E-mail:[email protected] (Dr. Hu Tang); Tel: +86 027 86711615; Fax: +86 027

86815916 1

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Abstract

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Cellulose-based composite macrogels made by cellulose fiber/cellulose nanofiber

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(CCNM) was used as an intestine delivery vehicle for probiotic. Cellulose nanofiber

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(CNF)

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(TEMPO)-mediated oxidation system, and the carboxyl groups in CNF acted as pore

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size and pH responsibility regulator in CCNMs to regulate the probiotics loading and

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controlled release property. The macrogel presented a porosity of 92.68% with a CNF

20

content of 90%, and the corresponding released viable Lactobacillus plantarum

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(L.plantarum) was up to 2.68 × 108 cfu/mL. The porous structure and high porosity

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benefited L.plantarum cells to infiltrate into the core of macrogels. In addition, the

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macrogels made with high contents of CNF showed sustainable release of

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L.plantarum cells and deliver enough viable cells to the desired region of intestine

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tracts. The porous cellulose macrogels prepared by a green and environmental

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friendly method are potential in application of fabricating targeted delivery vehicles of

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bioactive agents.

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was

prepared

by

a

2,2,6,6-tetramethylpiperidine-1-oxyl

radical

Key words: probiotic; cellulose macrogels; pH-responsive; controlled release

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Introduction

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Probiotics, defined as living microorganism by the Food and Agricultural

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Association of the United Nations (FAO) and the World Health Organization (WHO),

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are able to supply the host healthful effects.1-2 Gradually increased researches of

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applying probiotics as health improver have been performed in the past years due to

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their advantageous health benefits.3 The delivery of probiotics to regulate

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gastrointestinal microbiome compositions show promising impact on treatments for

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various human diseases such as inflammatory bowel diseases,4 obesity,5 and diabetes,6

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etc. However, the viability of the probiotics is greatly affected by the surrounding

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environment such as processing, storage and human gastrointestinal tract. Especially,

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if the target is to deliver adequate viable probiotics to the small intestine, it is essential

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to create a protective carrier for probiotics delivery.1,

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technology to protect probiotics avoid the injury of the adverse conditions has become

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a hot spot.8-9 Polysaccharides (such as gum chitosan, pectin, and alginate) and

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proteins (gelatin, whey protein,) are two kinds of most commonly applied

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biopolymers for the encapsulation of probiotics.10-11 Only alginate possesses

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pH-responsive properties among them, however, the application of alginate is limited

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because of its poor supporting property as encapsulating material.12-13

7

The use of microcapsule

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Cellulose, which is the most abundant renewable natural polymer, has been widely

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used in many fields because of its biocompatibility and biodegradebility.14-15 Various

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cellulose derivatives such as hydroxypropyl cellulose and carboxymethyl cellulose

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have been extensively used as encapsulates.16-17 However, these cellulose derivatives 3

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are water-soluble and can be physical degraded. Therefore, these derivatives are not

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feasible for probiotics delivery in the small intestines.18 An ideal alternative solution

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is the use of native cellulose, which is a dietary fiber that cannot be digested, for

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delivering probiotics to the intestines.19-21 According to previous studies, regenerated

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cellulose macrogels could be prepared through a simple and green method by

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dissolving cellulose in alkaline/urea aqueous solutions at low temperature. The

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obtained regenerated cellulose macrogels have porous structures and can be used as

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scaffolds to load probiotics.22 However, without the protection of an outer shell, the

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probiotics could be easily released through the porous structure and killed by the

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simulated gastric fluid.

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To tackle this issue, an effective method was to introduce carboxylic groups onto

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the

cellulose

surface

by

2,2,6,6-tetramethylpiperidine-1-oxyl

radical

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(TEMPO)-mediated oxidation system.23 This oxidation protocol can convert primary

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OH groups in anhydroglucose units to carboxylic moieties successfully.24 A large

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amount of carboxyl groups can provide cellulose-based materials with pH-responsive

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properties. In addition, the electrostatic repulsions caused by the carboxyl anions

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(COO-) in TEMPO oxidized cellulose nanofibers (CNF) can increase the space in the

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networks and control the pore size.25

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Here, we have shown pH-responsive porous cellulose-based composite macrogels

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with controlled pore size fabricated by cellulose fiber and cellulose nanofiber with a

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green and simple method. The morphology, structure, porosity and swelling behavior

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of the macrogels were investigated. Meanwhile, these macrogels were applied in 4

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loading and release of probiotics to evaluate the probiotics distribution and the release

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properties of the macrogels. Hence, the pH-responsive and pore size controllable

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cellulose-based macrogels can be used as potential encapsulation for the protection

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and delivery of probiotics.

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Materials and methods

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Materials

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Native cellulose (α-cellulose > 95%) was obtained from Hubei Chemical Fiber Co.,

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Ltd. (Xiangfan, China). An Ubbelohde viscometer was used to measure the

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viscosity-average molecular weight (Mη) of cellulose and the Mη was calculated

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according to the Mark-Houwink equation ([η] = 3.72 × 10-2 Mη0.77),26 to be 6.2×104

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Da. 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), sodium bromide (NaBr) and

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sodium hypochlorite (NaClO) were acquired from Sigma-Aldrich, USA. Pepsin

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(1:10000) and trypsin (1:250) were purchased from Biosharp Co., Ltd, China. The

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activity of trypsin and pepsin was 250 and 3000 u/mg, respectively. Other chemicals

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at analytical grade were obtained from Sinopharm Chemical Reagent Co., Ltd.

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(Shanghai, China). Lactobacillus plantarumsubsp. Plantarum CICC 6240 used as

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type strain was supplied by the China Center of Industrial Culture Collection.

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Preparation of CNF

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The native cellulose was modified through a TEMPO-mediated oxidation reaction

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as previously reported by Saito et al.23 Typically, the cellulose fibers were suspended

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in water at a concentration of 1 wt% with the addition of TEMPO (16 mg/g cellulose)

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and sodium bromide (100 mg/g cellulose). NaClO (7.5 mmol/g cellulose) was added 5

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dropwise to the suspension with continuous stirring at 500 rpm. The pH was

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maintained at 10 by adding 0.1 M NaOH until all NaClO was consumed. Deionized

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water was used to wash the CNF thoroughly by filtration and the CNF was collected

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when the filtrated solution reached the neutral point and the CNF was stored at 4 ℃

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for further use.

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Preparation of Cellulose-based Composite Macrogels

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The preparation of cellulose-based composite macrogels from cellulose fiber and

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cellulose nanofiber (CCNM) was shown in Figure 1. Briefly, 4 wt% of cellulose fiber

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(CF) and freeze-dried CNF were added into an aqueous LiOH/urea/water (8.7/12/79.3)

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solution and store in the fridge at -20 ℃. Then CF and CNF were dissolved with a

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freezing-thawing method.22, 27 The resultant composite solution was centrifuged at

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8230 g by Avantj J-25 (Beckman Coulter, America) for 20 min at 4 ℃ for

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degasification. The obtained composite solution was dropwise extruded with a syringe

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into dilute hydrochloric acid (10%) under constant magnetic stirring, and the resultant

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macrogels were washed thoroughly with deionized water until no inorganic salt and

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HCl exist. A series of macrogels were obtained by verifying the weight ratio of CF

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and CNF (10:0, 7:3, 5:5, 3:7 and 1:9), and these macrogels were coded as CFM,

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CCNM-1, CCNM-2, CCNM-3 and CCNM-4, respectively. Finally, the macrogels

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were lyophilized for characterization and encapsulation experiments.

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Encapsulation of Probiotic in Macrogels.

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L.plantarum was selected as the model probiotic throughout the experiments. A

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total of 0.1 g of sterilized lyophilized macrogels was immersed in 80 g of sterile De 6

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Man, Rogosa and sharp (MRS) medium,12 and then 1 mL 1×107 cfu/mL of

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L.plantarum strain was inoculated. The L.plantarum encapsulation macrogels were

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separated from the MRS broth after cultivation at 30 ℃ for 24 h.

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Characterization.

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The carboxyl contents of CNF samples were determined by conductometric

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titration method.28 Typically, 0.1 g CNF was dispersed in 100 mL deionized water and

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0.1 M HCl was used to adjust the pH of the suspension to 2.5. The suspension was

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titrated with 0.01 M standardized NaOH by adding 0.2 mL aliquots in 20 second

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intervals until the pH value reached 11, and the conductivity was measured during the

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process with a conductometric station (Seven Compact, Mettler-Toledo). The

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carboxyl contents of CNF were calculated as 1.43 ± 0.09 mmolg-1.

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The macro structures of the CFMs and CCNMs were photographed by using a

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digital camera (Nikon D3400, Japan). Fourier transform infrared spectroscopy (FT-IR)

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was recorded by a Fourier transform infrared spectrometer (1600, PerkinElmer Co.,

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USA). All spectra were recorded between 4000 and 400 cm-1, with a resolution of 4

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cm-1 and 16 scans. A wide-angle X-ray diffraction (XRD) was measured by XRD

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diffractometer (X’ Pert Pro, PANalytical, NL). And the patterns were recorded from

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2θ = 5° to 50°, (steps of 0.05°, step time of 10 s), with Cu-Kα radiate at 40 kV and 30

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mA and monochromatized using a 20 µm Ni filter. Thermal gravimetric analysis

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(TGA) was performed using a thermogravimetric analyzer (Perkin-Elmer Co., USA).

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The samples were heated from 30 to 600 ℃ at a rate of 15 ℃ min-1 in nitrogen

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atmosphere. All the samples were abraded into powders and dried out at 60 ℃ for 48 7

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h and then subjected to FT-IR, XRD and TGA measurements.

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The surface morphology and microporous structure of these macrogels were

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observed with a scanning electron microscope (SEM, Hitachi, S-570). The macrogels

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were frozen in liquid nitrogen, lyophilized, and sputtered with gold prior to the SEM

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observation. The ImageJ software was used to analysis the average pore sizes from

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SEM images.29 Water saturation and immersion technique was applied to measure the

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porosity of macrogels. The pore volume was calculated from the diversity of weight

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between the dry stated and the fully saturated.30

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The swelling behavior of CFMs and CCNMs was investigated by placing these

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macrogels in simulated gastric fluid (SGF) for 2 h and then shifting them into

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simulated intestinal fluid (SIF) for 6 h. SGF was prepared by dissolution of 9 g of

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NaCl and 3.2 g of pepsin in 1000 mL of sterile water and the pH was adjusted to 1.2

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by 1 M HCl solution. 6.8 g/L KH2PO4 and 10 g/L trypsin were used to prepared SIF,

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and the pH was adjusted to 7.5 ± 0.1.22 Stereoscopic microscope (SZX16, Olympus

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Co., JP) was used to observe and record the whole process.

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Hoechst 33258 was used to stain the L.Plantarum cells encapsulated macrogels to

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investigate the distribution of L.plantarum cells in macrogels.13 The mixture was

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stored at 4 ℃ for 20 min then washed with the pure water to remove the free dyes

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prior. The macrogels were surrounded and covered by the frozen sliced embedding

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agent and cut by cryogenic cryotomy machine ( LEICA CM1850, LEICA, GER) into

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pieces at -35 ℃. The cross-section of macrogels was observed by fluorescent

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inverted microscope (IX71, Olympus Co, JP) to study the distribution of L.plantarum 8

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cells in macrogels. The L.plantarum strain presents blue fluorescence. The excitation

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and emission wavelength was 346 and 460 nm, respectively.

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Release behavior of L.plantarum from the macrogels

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The release behavior of L.plantarum cells from CFMs and CCNMs was

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investigated in SGF and SIF. The lyophilized CFMs and CCNMs (0.1 g) encapsulated

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with L.plantarum cells were immersed in SGF (10 mL) at 37 ℃ for 2 h with

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continuous oscillation at 140 rpm. Then, the macrogels were quickly shifted from

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SGF to SIF, and the survival and release assays in SIF were carried out at similar

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conditions. Aliquots (0.1 mL) were removed from the medium and serially diluted for

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5 times. 22 The viable counts were performed on MRS ager plates.

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Results And Discussion

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Figure S1 showed the FT-IR spectra of CF (a), CNF (b), CFM (c), CCNM-1 (d),

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CCNM-2 (e), CCNM-3 (f), and CCNM-4 (g), respectively. The broad absorption band

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at ~3400 cm-1 and the slight band at ~2900 cm-1 can be assigned to the stretching

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vibration of -OH and -CH/-CH2 groups on the sugar ring from cellulose,

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respectively.31 In the region between 1500 and 1300 cm-1, the absorption peaks were

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assigned as multiple vibration modes including -CH (-CH and -CH2 groups) and -OH

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bending. The strong bands in the range of 1200-1000 cm−1 were assigned as the

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stretching vibration of C-O from CH-OH and CH2-OH of the sugar ring. Compared

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with Figure S1a, the FT-IR spectrum of CNF in Figure S1b exhibited the similar

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profile but a new band was clearly visible at around 1731 cm-1, characteristic of

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carboxyl groups (COO-) in acidic form,31 indicating a successful modification of CF. 9

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It was clearly observed that the intensity of C=O stretching bands was gradually

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increased from Figure S1d-g due to the introduction of increased amount of carboxyl

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groups, and the absorption band was 1720 cm-1 1728 cm-1, 1730 cm-1and 1731 cm-1,

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respectively, indicated an increased hydrogen bond interaction between –OH and –

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COO-. 32

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The degree of crystallinity and crystalline structure of CF (a), CNF (b) and

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macrogels (c-g) were determined by X-ray diffraction analysis, and the results were

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showed in Figure S2. The diffraction pattern of CF and CNF showed peaks at 2θ =

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14.9° (101), 16.6° (10 1), 22.8° (002), 34.5° (040) corresponding to the typical

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cellulose I structure.33 This fact indicated that the crystals of cellulose did not change

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after oxidation. It is further stated that TEMPO oxidation was mainly in the

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amorphous region of the cellulose and the crystal form of the crystalline region, rather

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than the interior of the crystallization zone. With respect to the macrogels, the

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characteristic peaks at 2θ = 12.1°, 19.8°, 22.6° were attributed to (101), (101), (002)

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crystallographic planes of cellulose II,34 respectively. The crystallinity of the

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regenerated macrogels was smaller than that of the CF and CNF, and these results

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showed that the macrogels have many amorphous regions, which was the result of the

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formation of the porous structure. The XRD results indicated that the crystal structure

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of cellulose shifted from cellulose I to celluloseⅡ during the process of dissolution

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and regeneration. Although the physical properties such as wet density, particle size

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and pore size of the macrogels from CFM to CCNM (1-4) were somewhat different,

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their chemical structures were almost unchanged.

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-

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The thermal stability of CF, CNF and macrogels (c-g) were accessed by TGA with

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a heating rate of 15 ℃/min ranging from 30 ℃ and 600 ℃ under nitrogen gas flow.

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As shown in Figure S3, a weight loss of 1 ~ 5% under 100 ℃ was observed for the

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samples, and it was probably the result of the loss of residual moisture from the

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samples.35 The thermal degradation initiation temperature (Td) of CNF was about

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220 ℃, which was much lower than that of CF (285 ℃). This may be due to the fact

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that a large number of C6 primary hydroxyl groups on the surface of the cellulose

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were oxidized to carboxyl groups and the carboxyl groups began to degrade and

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volatilized at lower temperatures, resulting in a significant decrease in thermal

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stability.36 In addition, the thermal degradation of CNF is resolved into two processes,

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including the main decomposition process at 200-350 ℃ and a slow charring process

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at 350-600 ℃.37 All samples exhibited good thermal stability up to about 200 ℃,

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and the macrogel products with the better heat resistance could be obtained when the

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content of cellulose was higher. Briefly, all macrogels have good thermal stability

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below 200 ℃, which was essential for practical application.

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In order to verify the pH-responsibility of the macrogels, the swelling behavior of

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macrogels in SGF and SIF was observed by stereoscopic microscope. CFM showed

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no difference under SGF and SIF (Figure 2). As for CCNM-1, the swelling effect

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hardly worked since there was not enough carboxyl groups introduced into the

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macrogels to respond to ambient pH. It was found that when the content of carboxyl

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groups increased, the CCNMs had slight shrinkage in SGF but apparently bulk up

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when transferred to SIF and the phenomenon was more obvious in CCNM-3 and 11

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CCNM-4. The phenomenon implied that CCNMs exhibited a pH-responsible

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behavior and it was positively correlated with the content of carboxyl groups

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introduced into the macrogels. The pH-responsive is important for the targeted release

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of probiotics in the gastrointestinal tract as well as enhancing the acid resistance of the

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macrogels.

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The SEM images of CFM, CCNM-1, CCNM-2, CCNM-3 and CCNM-4 were

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shown in Figure 3. The macrogels presented three-dimensional macro-porous inner

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structures. The porous structure was resulted from the H2O-played an important role

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in inducing phase separation during the sol−gel process, where the regions of

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solvent-rich lead to the formation of pores.25, 38-39 The average pore size of macrogels

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was 0.60 µm, 1.08 µm, 1.45 µm, 1.74 µm and 2.11 µm with the gradual increments

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usage of CNF in the macrogels. The absorbed water which worked as pore former

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resulted in a more loose structure. The results indicated that the introduced carboxyl

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groups worked as a pore size regulator, whereas the cellulose backbone played the

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role of strengthening the porous structure.25 Thus, a large amount of water molecules

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could be easily assimilated and spread into macrogels, increasing the swelling ratio

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and the pore size.25 It was worth mentioning that the larger pore size structure was

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convenient for probiotics to spread into the core of macrogels and can also help to

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enhance the loading capacity of probiotics.

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The ability of loading L.plantarum cells in macrogels was confirmed by observing

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the macrogels through the SEM. It could be easily observed from the SEM images in

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Figure 4 that a large number of L.plantarum cells were densely distributed on the 12

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surface of CFMs and CCNM-1 to CCNM-3, while L.plantarum cells were evenly

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spread on the pore walls of CCNM-4. It showed that the difference of pore size of

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macrogels leads to the different ability of loading L.plantarum cells. The distribution

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of L.plantarum cells in the macrogels was further verified by staining the macrogels

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containing L.plantarum cells with Hoechst 33258 and observing the cross-section

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using fluorescent inverted microscope. As shown in the right side of Figure 4, the

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fluorescence intensity of Figure 4a-d was gradually increased and mainly

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concentrated in the outer edge of the macrogels and had a tendency to gradually

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spread to the inside. In contrast, the fluorescence image of Figure 4e showed a strong

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fluorescence in the core of CCNM-4, indicating that L.plantarum cells had penetrated

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deep into the core of CCNM-4. Accordingly, the difference in pore size affected the

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distribution of cells in macrogels greatly. The larger pore size was more suitable for

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L.plantarum cells to spread into the macrogels, thus contributing to effective loading

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of probiotics.

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The total number of the encapsulated L.plantarum cells in macrogels and the

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porosity of macrogels had been measured. The encapsulated L.plantarum cells were

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reflected by the amount of cells released from macrogels in SIF without immersed in

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SGF, because of the insolubility of cellulose in water. The relationship between the

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encapsulated L.plantarum cells and the porosity was displayed in Figure 5a. The

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Figure 5b showed the release files of viable cells from macrogels in SIF. It was

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clearly observed that the encapsulated L.Plantarum cells were positively correlated

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with the porosity. The porosity of macrogels was gradually increased from 78.19% to 13

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92.68% with the increments of carboxyl groups introduced during making macrogels,

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and the encapsulated L.plantarum cells grew from 9.67 × 107 cfu/mL to 2.68 × 108

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cfu/mL according to the same trend. It was concluded from Figure 4 and Figure 5 that

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the pore size and porosity had significant impacts on the loading and release of viable

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cells. The larger pore size and higher porosity were more helpful for the L.plantarum

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cells to colonize the interior of the macrogels, which indicated that manipulating the

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microstructure of the cellulose macrogels could control the loading of probiotic cells.

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In order to verify the protective and sustained release effect of macrogels on

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probiotics, the macrogels loaded with L.plantarum cells were immersed in SGF for 2

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hours and then transferred to SIF for simulated release. After exposed to SGF for 2 h,

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the release amount and trend of viable L.plantarum cells from macrogels presented a

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distinct difference as shown in Figure 6. The released amount of viable L.plantarum

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cells from CFM, CCNM-1, CCNM-2, CCNM-3, CCNM-4 were 5.33 × 106 cfu/mL,

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7.77 × 106 cfu/mL, 9.98 × 106 cfu/mL, 1.70 × 107 cfu/mL, 2.23 × 107 cfu/mL,

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respectively. It was obvious that the total amount of released L.plantarum cells

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gradually increased.

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The L.plantarum cells were liberated from all the samples and exhibited linear

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growth within the first 10 min. However, the release rate of CFM, CCNM-1 and

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CCNM-2 was significantly reduced after 30 minutes and the release amount of

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L.plantarum cells reached the maximum at about 180 min. This was because that the

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probiotic cells were mainly distributed on the surface of CFM, CCNM-1 and

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CCNM-2 which echoed with the results of the fluorescence uptake microscopy. In the 14

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contrast, CCNM-3 and CCNM-4 showed a more sustained release of viable

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L.plantarum cells and the cells in CCNM-3 and CCNM-4 kept a slow release.

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However, L.plantarum cells were fully released from CCNM-3 at 240 min, while the

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maximum release degree of CCNM-4 was at 360 min. The results manifested

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CCNM-4 is helpful to provide better shelter for L.plantarum cells and longer duration

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release of viable cells in SIF owing to the porous structure and rigid supporting

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property. The results of fluorescence uptake microscopy already showed that the

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probiotic cells have infiltrated into the core of the CCNM-4 (Figure 4) which means

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the release of L.plantarum cells was in a sustainable state. Initially there was a burst

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release because of the emancipation of the outermost cells in CCNM-4. Then it was

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followed by a decreased releasing of inside L.plantarum cells. The main reasons for

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the slow release of CCNM-4 were as follows: CCNM-4 contains a large amount of

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carboxyl groups, giving CCNM-4 a certain pH response, so that CCNM-4 showed a

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certain contraction effect in the SGF; on the other hand, cellulose can’t be dissolved

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or digeated; therefore, the release of L.plantarum cells in CCNM-4 was in a

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sustainable way rather than in a burst way, which could be helpful to ensure that a

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sufficient amount of L.plantarum cells reaches the desirable region.

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It was displayed that the maximum viable L .plantarum cells released from

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CCNM-4 reached 2.23 × 107 cfu/mL, while studies have reported that the minimum

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intake of probiotics must reach 106 ~ 107 cfu/mL in order to give full play to its

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probiotic function.40 And the result indicated that CCNM-4 can transport enough

315

probiotics to the intestine to play its function. 15

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In this work, CCNM was successfully created by dissolving a mixture of CF and

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CNF prepared from a TEMPO mediated system in an aqueous LiOH/urea solution

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and extruded into macrogels with an extrusion dropping technology. In the composite

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macrogels, the carboxyl groups in CNF worked as a pore size regulator, whereas the

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cellulose backbone played the role of strengthening the porous structure. Release and

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distribution of L.plantarum cells encapsulated in the macrogels, were determined by

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the porosity and the pore size. The loading capacity of the blended macrogels

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increased with the increase of the pore size, and the macrogels with larger pore size

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showed better sustained release effect, as the maximum viable released cells of

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CCNM-4 was more than 2.23 × 107 cfu/mL. The results of SEM and fluorescence

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microscope indicated that CCNM-4 provided the desirable conditions for spreading

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L.plantarum cells to the core of macrogels, while small-pore-macrogels prevented the

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spread of probiotics into macrogels. In conclusion, the carboxyl groups in CNF can be

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used to adjust the pore size of cellulose macrogels to improve their loading capacity

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and the cellulose scaffold can be used as a suitable medium to deliver the nutrient

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stably and is promising for application in the biopharmaceutical industry.

332 333

Supporting Information

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FT-IR spectra (Figure S1) and X-ray diffraction profiles (Figure S2) of CF (a), CNF

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(b), CFM (c), CCNM-1 (d), CCNM-2 (e), CCNM-3 (f), and CCNM-4 (g); Thermal

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analysis of CF, CNF, CFM, CCNM-1,CCNM-2,CCNM-3 and CCNM-4 (Figure S3)

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Funding

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This work was supported by the National Natural Science Foundation of China

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(31701560, 31671820 and 31501423), the Basic Applied Research Project of Wuhan

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City (2016020101010095), Agricultural Science and Technology Innovation Project

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of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-OCRI) and the

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Director Fund of Oil Crops Research Institute (1610172014006).

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Notes

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The authors declare no competing financial interest.

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FIGURE CAPTIONS.

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Figure 1. Scheme of TEMPO-mediated oxidation of cellulose (a), scheme of

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preparation of CCNM, and swelling mechanism of CFM and CCNM (c).

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Figure 2. The images of CFM, CCNM-1, CCNM-2, CCNM-3 and CCNM-4 in their

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dry states and swelling states in SGF and SIF at different time.

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Figure 3. SEM images (left) and pore size distribution (right) of CFM (a), CCNM-1

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(b), CCNM-2 (c), CCNM-3 (d) and CCNM-4 (e).

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Figure 4. SEM images (left) and the cross-sectional fluorescence images (right) of

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CFM (a), CCNM-1 (b), CCNM-2 (c), CCNM-3 (d) and CCNM-4 (e) with

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encapsulated L.plantarum.

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Figure 5. The relationship between released viable cell count and porosity of

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macrogels (a) and release files of viable cells from macrogels in SIF (pH 7.5) (b).

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Figure 6. Release files of viable cells from macrogels in SIF (pH 7.5) after immersing

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in SGF for 2 h (pH 1.2).

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Table of Contents Graphics

This work uses a green and environmental friendly method to prepare porous cellulose macrogels that can be applied to targeted delivery probiotics.

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