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

1

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

5

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

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*

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

86815916 1

ACS Paragon Plus Environment


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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

16

(CNF)

17

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

18

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

19

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

21

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

22

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

23

macrogels made with high contents of CNF showed sustainable release of

24

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

25

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

26

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

27

bioactive agents.

28

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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2


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Introduction

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Probiotics, defined as living microorganism by the Food and Agricultural

32

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

35

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

36

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

39

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

40

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

41

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

47

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

49

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

65

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

67

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

73

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

84

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

93

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

94

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

95

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

98

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

99

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

100

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)

105

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

106

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

107

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

108

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

109

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

110

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,

113

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

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were lyophilized for characterization and encapsulation experiments.

115

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

123

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

126

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

127

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.

129

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

130

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

131

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

132

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

134

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

135

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

136

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

137

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

138

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

139

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

144

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

154

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

158

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

159

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

170

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

171

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,

177

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

178

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

179

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

180

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

181

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

185

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

194

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

196

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

200

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

201

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

202

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

203

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,

205

their chemical structures were almost unchanged.

-

-

10


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

207

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

209

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

210

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

215

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

220

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

222

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

224

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

225

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

226

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

227

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

229

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.

233

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

235

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

240

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

242

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

243

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

244

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

245

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

248

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

249

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

251

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

254

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

255

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

256

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

258

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

259

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

261

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

263

of probiotics.

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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



272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

283

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

284

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

285

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

286

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

287

gradually increased.

288

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

289

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

290

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

291

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

292

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

293

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

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

311

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

312

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

313

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

314

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

315

probiotics to the intestine to play its function. 15



316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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

328

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

329

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

330

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

331

stably and is promising for application in the biopharmaceutical industry.

332 333

Supporting Information

334

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

335

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

336

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

337 16


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Funding

339

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

340

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

341

City (2016020101010095), Agricultural Science and Technology Innovation Project

342

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

343

Director Fund of Oil Crops Research Institute (1610172014006).

344

Notes

345

The authors declare no competing financial interest.

346

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467

FIGURE CAPTIONS.

468

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

469

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

470

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

471

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

472

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

473

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

474

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

475

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

476

encapsulated L.plantarum.

477

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

478

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

479

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

480

in SGF for 2 h (pH 1.2).

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Figure 1.

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485 486

Figure 2.

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Figure 3.

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491 492

Figure 4.

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Figure 5.

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497 498

Figure 6.

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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.

31


Cellulose-based composite macrogels from cellulose fiber and

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