Porous Cellulose Microgel Particle: A Fascinating Host for the

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Porous cellulose microgel particle: A fascinating host for the encapsulation, protection and delivery of Lactobacillus plantarum Wei Li, Xiaogang Luo, Rong Song, Ya Zhu, Bin Li, and Shilin Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00481 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 14, 2016

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Journal of Agricultural and Food Chemistry

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Porous cellulose microgel particle: A fascinating host for the encapsulation,

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protection and delivery of Lactobacillus plantarum

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Wei Lia, Xiaogang Luob, Rong Songa, Ya Zhua, Bin Lia, Shilin Liua*

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a. College of Food Science & Technology, Huazhong Agricultural University, Wuhan,

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Hubei, 430070, China;

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b. Key Laboratory of Green Chemical Process of Ministry of Education, School of

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Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan, Hubei,

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430073, China.

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*Corresponding Author: College of Food Science & Technology, Huazhong

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Agricultural University, Wuhan, Hubei, 430070, China Tel: +86-027-87282111; Fax:

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+86-027-87282111; E-mail: [email protected] (S. Liu)

ACS Paragon Plus Environment


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Abstract

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Advances in probiotic markets are always restrained by low viable loading capacity

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and poor viability. Herein, cellulose microgels (CM) with high porosity of

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95.83±0.38 %, prepared by sol-gel transition method, turned out to be a hospitable

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host that accommodated a large number of viable L. plantarum higher than 109 cfu/g.

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The unique porous structure fascinated probiotics to penetrate deep inside the core of

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microgels. The conjugation with alginate helped to better acid resistance and bacterial

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survival of the probiotics. When compared with Ca-alginate gels, cores-shell gels

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showed sustainable release of L. plantarum cells without damage of viability lasting

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for 360 min in simulated intestine fluid. Cellulose ingredient helped to sustaining the

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viable cell release for longer duration and affording better shelter for L. plantarum

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cells due to the porous structure and rigid supporting property. The cores-shell gels

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are promising for constructing targeted delivery vehicles of bioactive nutrients.

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Keywords: cellulose, microgels, cores-shell, probiotic

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Introduction

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Probiotics are microorganisms that are believed to provide health benefits when

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administered in adequate amounts. 1-4 The concerns throughout the scientific research

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reside in the low loading capacity and viability during use and storage, and finally the

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ability to survive in stomach acids.

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increase the loading capacity and improve the viability of probiotics, major emphasis

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have been given to protect them with the help of various encapsulating materials,

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including alginate,

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and starch

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the manufacture of stirred yogurts to prevent syneresis.

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material, for example alginate, has limited loading capacity due to its poor supporting

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property. The materials except alginate have the disadvantage of no response to pH

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changing. A comprehensive selection of materials with stronger stability against

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digestion plus higher loading capacity is critical for escorting adequate number of

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alive probiotics to the desired place in human body.

15

9

chitosan,

10

5-8

Several attempts have been performed to

carrageenan,

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gums,

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gelatin,

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whey protein,

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etc. Some of them could be lyophilized protective or food stabilizers in 16

Single encapsulating

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Cellulose, the most abundant polysaccharide on earth, belongs to the family of

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dietary fiber with strong hydrophilic bulking ability which is good for human intestine

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health.17-21

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carboxymethyl cellulose, are widely applied as encapsulants.

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water-solubility of cellulose derivatives leads to physical degradation such as loss of

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mechanical properties and fiber embrittlement, which makes it impossible to be

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scaffolds with rigid supporting property. Native cellulose is unable to be dissolved in

Cellulose

derivatives,

including

hydroxypropyl


cellulose 22, 23

and

But the


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normal solvents and digested by human body, so it can provide more stable

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environment for probiotics when passing through the gastrointestinal tract. However,

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probiotic products based on native cellulose has been rarely reported, mainly owing to

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the difficulty in processing native cellulose polysaccharide via common solvent and

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conditions.24 We have put an intensive research on the construction of functional

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materials based on cellulose by using a series of developed solvents.

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previous work, alkaline/urea aqueous solution at low temperature turns out to be the

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most green and facile approach to dissolving cellulose, and the solvents are very easy

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to be removed after the regeneration process. The obtained cellulose microgels with

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porous structure are used as scaffolds to synthesize inorganic nanoparticles or curable

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organic prepolymers in situ for the construction of the functional materials,27,

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indicating the cellulose gel based scaffold is a novel support for the incorporated

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components. Inspired by these results, we attempt to encapsulate probiotics into the

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porous cellulose microgels in this work.

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In our

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The main objective of this work relied on the improvement of probiotic viability

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and providing a strategy to prolong the release time so that probiotic can reach the

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desire region in human body, large intestine. Porous matrix usually outperforms one at

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a planar surface by the virtues of larger surface area and open structure that aid

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penetration, which can efficiently raise probiotic loading capacity and afford better

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shelter for probiotic residence.

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tracts, there is an appreciation of developing the formula in conjugation with a

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pH-trigger. The performance of probiotic encapsulation by using porous cellulose

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Given the complex physiology of gastrointestinal


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microgels with and without modification of alginate has also been compared. An

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understanding of alginate available to probiotic proliferation was further exploited by

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modulating the proportion of ingredients in cellulose-alginate composite microgels.

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We also highlighted the inherent strong hydrophility of cellulose for absorbing

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sufficient culture medium, which was conducive to high viability of probiotic cells.

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Modified porous cellulose gels with pH-responsibility appear to be the most

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promising candidate for encapsulation and intelligent release of bioactive drug and

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bio-nutrients.

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Experimental

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Materials. Native cellulose (α-cellulose ≥ 95%) was obtained from Hubei Chemical

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Fiber Co. Ltd. (Xiangfan, China). Its viscosity-average molecular weight (Mη) was

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about 1.07 × 105 in cadoxen at 25 oC and the calculation relied on the Mark-Houwink

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equation. Sodium alginate (ALG), paraffin oil and pepsin were purchased from

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Aladdin Reagent Database Co. (Shanghai, China). Trypsin (1: 250) was obtained from

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Biosharp Co., Ltd, China. The activity of Trypsin and pepsin is 250 U/mg and 2500

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U/mg, respectivity. Tween 80 (Cat. No. 30189828) and Span 80 (Cat. No. 30170828)

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

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(Shanghai, China). Hoechst 33342 Staining Kit was supplied by Biyuntian

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Biotechnological Institute. Man, Rogosa and Sharpe (MRS) broth and agar were from

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Qingdao Hope Biol-Technology Co., Ltd. Other chemicals at analytical grade were

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supplied by the Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The type

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strain of Lactobacillus plantarum subsp. plantarum CICC 6240 was bought from



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China Center of Industrial Culture Collection.

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Preparation of Regenerated Cellulose-Alginate Microgels. The freezing-thawing

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method was used for the preparation of cellulose solution.

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composite solution was obtained by dissolving sodium alginate (4.3 wt%) into

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aqueous alkaline/urea solution and subsequently mixed with cellulose solution in

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different proportions. The resultant cellulose solutions or cellulose-alginate solutions

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were subjected to be centrifuged at 9418 g at 4 oC for 8 min to eliminate bubbles in

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the viscous solution. Then 40 mL of the cellulose solution or cellulose-alginate

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solution were dropped into a well-mixed suspension containing 200 mL of paraffin

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oils, 3g of Span 80 and 1 g of Tween 80 under vigorous but well-proportioned

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mechanical stirring for 3 h, dilute hydrochloric acid (10%) was subsequently added

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until the pH of resultant suspension reached 7, then the regenerated cellulose

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microgels (CMs) or cellulose/alginate microgels (CAMs) were obtained. The resulting

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CAMs were named as CAM-1, CAM-2, CAM-3 and CAM-4, according to the

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cellulose:alginate volume ratio of 4:1, 3:2, 2:3 and 1:4. After the removal of liquid

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paraffin, CAMs were immersed in 0.05 M CaCl2 solution under gentle mechanical

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stirring for 30 min. Then CMs and CAMs were washed thoroughly with deionized

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water and hot ethanol, and there was no inorganic salt or surfactant existing in the

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obtained microgels. Finally, the clean microgels were lyophilized.

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Encapsulation of Probiotic in the Microgels. Lactobacillus plantarum (L.

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plantarum) was used as the representative probiotic and kept in cryovials at -80 oC

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before using. Each cryovial contained 0.5 mL of glycerol, 0.5 mL of fresh aseptic


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The cellulose-alginate

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MRS broth and 1 mL of L. plantarum strain at logarithmic phase. 0.1 g of lyophilized

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microgels (CMs and CAMs) were sterilized at 100 oC for 20 mins, then immersed in

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100 mL aseptic MRS broth, and subsequently inoculated with 1 mL of L. plantarum

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strain from cryovial. After cultivation at 30 oC for 24 h, the encapsulation process was

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finished by separating L. plantarum-encapsulated-microgels from the culture medium

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via mesh screen.

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Preparation of Cores-shell gels. Firstly, 0.1 g of lyophilized CMs were used to

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encapsulate L. plantarum, the encapsulation process was described above, after that,

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the L. plantarum-encapsulated-CMs were evenly dispersed in 20 mL of 5% sterile

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sodium alginate solution under gentle magnetic stirring. Then the resultant mixture

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was dropwise extruded by using a syringe and a pump (flow rate 17.6 mL/h) into

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CaCl2 (0.05 M, 50 mL) solution under vigorous and uniform magnetic stirring for 30

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min, and the cores-shell gels were obtained. Ca-alginate gels as the control group in

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the release test were prepared by extrusion technique. 20 mL of sterile sodium

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alginate solution was mixed with 2 mL cell culture that was cultivated at 30 oC for 24

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h, the final concentration of sodium alginate was 5%, the mixture was extruded into

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CaCl2 (0.05 M, 50 mL) solution under vigorous and uniform magnetic stirring for 30

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min. Finally, the resultant Ca-alginate gels and cores-shell gels encapsulated with L.

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plantarum cells were separated by mesh screen.

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Characterization. The surface morphology of CMs and CAMs were characterized by

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using scanning electron microscope (SEM, TM3030, Hitachi Ltd., Japan). Wide-angle

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X-ray diffraction (XRD) measurement was carried out on a XRD diffractometer



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(D8-Advance, Bruker, USA). Samples were ground into powders and dried in vacuum

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oven at 60 ºC for 48 h. The patterns with Cu Kα radiation at 40 kV and 30 mA were

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recorded in the region of 2θ from 5 to 80o. Thermal gravimetric analysis (TGA) was

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performed by using a thermogravimetric analysis (Netzsch, German). About 5 mg of

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the tested samples were placed in a platinum pan and the test temperature ranged from

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303 to 863 K at a rate of 10 K•min-1 in N2 atmosphere. The dry powder samples were

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mixed with defined amount of KBr and pressed for the FT-IR tests. Fourier transform

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infrared (FT-IR) spectra (170-SX, Thermo Nicolet Ltd., USA) were recorded in the

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wavenumber range from 4000 to 400 cm-1 and at a resolution of 1.928 cm−1. The

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number of FT-IR scans was 64 times. The porosity of microgels was measured by the

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water saturation and immersion technique, which was conducted by saturating a

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sample with water, and calculating the pore volume from the weight difference

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between the fully saturated and dry states. 31

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The CAMs encapsulated with L. plantarum cells were stained with Hoechst 33258

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staining solution at 4 oC for 20 mins and then washed with pure water to remove free

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dyes according to the manufacturer’s instructions (Beyotime). With respect to

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cores-shell gels, CMs were dyed according to the above-mentioned process prior to

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the preparation of cores-shell gels. The distribution of L. plantarum cells in microgels

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and cores-shell gels was investigated with the fluorescence microscope (Nikon 80i,

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Japan) and the confocal microscope (Olympus FV1200, Japan). The confocal

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scanning was kept adjusting until the largest fluorescent cross-section in the microgels

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could be observed and then pictured. The L. plantarum strain presented blue


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fluorescence. The excitation wavelength and emission wavelength was 346 nm and

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460 nm, respectively.

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Swelling Behavior of Composite Microgels. Swelling behaviors of CMs and CAMs

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in mediums with different pH were investigated by static micro particle image

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analyzers (Winner 99D, China). Hydrochloric acid and sodium hydroxide were used

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to prepare five buffer solutions with the pH of 1, 3, 6, 8 and 10. A microgel particle of

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each sample was selected to be immersed in the buffer (pH 1) at first and then

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transferred to the next buffer in the order of pH valve (low to high). The immersion in

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each buffer lasted for 2 h and the gel particle was taken photos by a camera in the

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micro particle image analyzer before it was transferred into next buffer. The

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experiment of each sample was repeated for three times.

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Release behavior of L. plantarum from the Microgels. The release behavior of L.

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plantarum cells from CMs, CAMs, Ca-alginate gels and cores-shell gels in simulated

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gastric fluid (SGF) and simulated intestinal fluid (SIF) were investigated. Simulated

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gastric fluid was prepared by dissolving 9 g NaCl in 1000 mL sterile water and

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adjusting pH to 1.2 with 1 M HCl solution, and then dissolving 3.2 g of pepsin in it.

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Simulated intestinal fluid was prepared by using 6.8 g/L KH2PO4, containing 10 g/L

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trypsin and the pH was adjusted to 7.5±0.1. Firstly, 0.1 g of lyophilized CAMs were

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used in encapsulation, and subsequently immersed in 10 mL of SGF, then

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immediately placed in an incubator shaker at 37 oC and 140 rpm. As for Ca-alginate

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gels and cores-shell gels, 1 g of wet gels was harnessed in the experiment. After 120

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min, aliquots (1 mL) taken from the SGF were enumerated by viable counts on MRS



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agar plates. Besides, the SGF were also examined under a microscope after staining

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with methylene blue solution. The survival and release assay in SIF were conducted at

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the similar conditions, and at certain time intervals, aliquots (1 mL) taken from the

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medium were enumerated by using tenfold serial dilution and viable counts on MRS

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agar plates.

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Statistical analysis. All experiments were conducted three times on freshly prepared

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samples. Results were reported as the averages and standard deviations of these

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measurements.

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Results and discussion

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The SEM images of CMs and CAMs are shown in Figure 1. Apparently, CMs

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exhibited good spherical shape with the diameter about 500 um and the surface

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presented highly crowded porous 3D structure. The porous structure was resulted

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from the gelation and regeneration process of cellulose solution when treated with a

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large volume of non-solvent and relatively high temperature, the phase separation

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happened during the process, and the solvent-rich regions in cellulose solution

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contributed to the pore formation. The obtained cellulose microgels had very high

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porosity of 95.83±0.38 %, and the SBET was about 109 m2•g-1. As for CAMs that were

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prepared with the similar condition, the microstructures were significantly different

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from that of the pure cellulose microgels. Composite microgels with lower content of

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alginate (CAM-1 and CAM-2) were round gel particle-like and had porous structure,

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however, the pore size was smaller than that of cellulose microgels, and the pore

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morphology changed from network-like to lamellar structures, which would be


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ascribed to the self-association between alginate macromolecules and interaction of

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alginate and cellulose macromolecules. With increasing the content of alginate in the

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composite microgels, there appeared obvious changes in the morphology and

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microstructure of CAM-3 and CAM-4, it became less porous but more compact. The

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appearance of CAM-4 was even collapsed and irregular, mainly owing to the weaker

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supporting force of alginate. The diffusion setting rule during regeneration process

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rendered a higher polymer concentration on the surface of microgels. When the

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microgels dried, the interior shrank to a larger extent than the exterior, and the

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mechanical supporting force of alginate was too weak to withstand the stress, thus

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generating wrinkled and uneven morphology with decreased size. Besides, when

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CAM-4 came back to water, it could become round microgel particle again (Figure S4

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e). Briefly, cellulose ingredient had an obvious influence on the morphology and

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microstructure of the composite microgels, so the microstructure could be controlled

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via changing the content of the incorporated cellulose macromolecules. The results of

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FT-IR and XRD demonstrated that alginate had been incorporated in the composite

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microgels, which was important for the preparation of biomacromolecule based

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microgels with the pH responsive properties (Figure S1 and S2). Besides, the thermal

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stability of the composite microgels was improved with increasing the content of the

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cellulose in the microgels, and all samples maintained stable below 200 oC, which

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was crucial for practical application for industry (Figure S3). The composite

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microgels presented pH-responsible behavior that was positively correlated to the

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content of alginate ingredient in the composites (Figure S4). Given the merits of



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porous structure and excellent pH-responsibility, the composite microgels were used

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to encapsulate L. plantarum cells. Obviously, there were plenty of L. plantarum cells

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on the surface of all microgels, manifesting the successful encapsulation of probiotics.

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Whereas, the distribution of L. plantarum cells in Figure 1f and g was much looser

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while bacteria almost accumulated to multilayers on the surface of CAM-4. Alginate

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is a kind of prebiotic that benefits the growth of bacteria,

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population could be observed with the increasing content of alginate in the CAMs. In

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addition, the pores were almost invisible and the main reasons were speculated as

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follows: firstly, both cellulose and alginate were highly hydrophilic polysaccharides,

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so the microgels could absorb sufficient MRS broth during the encapsulation, which

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formed thin layers after the drying process that might block the aperture gaps in pores.

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Secondly, plenty of bacteria were embedded in the cavities of microgels. The

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accumulation of L. plantarum cells fully utilized the advantage of porous structure,

249

such as, the open structure, larger surface areas.

32

hence larger cell

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To investigate whether L. plantarum cells penetrate into the microgels, the novel

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confocal laser scanning microscopy-based method was used to image the distribution

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of L. plantarum cells in the cross-section of the microgels (Figure 2). There appeared

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some blue hollow shapes in the merged image of CAM-4, while in that of CAM-1, the

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blue circles was solid, indicating the penetration of probiotic cells was deep enough to

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the core of CAM-1, but as for CAM-4, the probiotic cells mostly concentrated on the

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surface instead of core. Accordingly, the difference in distribution of cells in

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microgles greatly relied on the pore structure and surface morphology. More porous


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scaffold led to the interconnected structure so that bacteria could penetrate deep into

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the microgels, thus contributing to effectively controlled release and affording better

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habitat for bacterial metabolism and proliferation.

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Due to the insolubility of cellulose, the difficulty in separation of the encapsulated

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bacteria from cellulose matrix made it impossible to measure the accurate amount of

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encapsulated L. plantarum, but it could be indirectly reflected by investigating the

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release behaviors in intestinal tract (Figure 3). There appeared obvious initial burst

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release of encapsulated bacteria from CAMs due to the presence of open pore

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structure and liberation of surface-bound bacteria. Little difference in amounts could

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be figured out between the released viable cells from CAMs. The released cells

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reached plateaus up to 107 cfu/mL in 30 mins. It should be noted that the loading

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amount of bacteria in the scaffold was never less than the maximum viable release

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count of L. plantarum. Take CAM-1 and CAM-3 for example, the encapsulated cell

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number must be higher than 6.7×109 and 7.8×109 cfu/g for the presence of hundred

272

fold dilution effect, respectively. In addition, we also investigated the survival and

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release behavior of composite microgels in simulated gastric fluids by means of

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dilution plate count, but it showed no viable cell in SGF. To further ensure whether

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there was L. plantarum cells releasing from the scaffold in SGF, the gastric solutions

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of all samples were examined under a microscope after staining with methylene blue

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solution (Figure S5 a-e). As we all know, dead bacteria cannot to reduce the oxidized

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methylene blue and the bacteria were stained blue. The released cells in SGF were

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blue and stationary, it indicated that the bacteria were unable to stand the high acid



280

environment and lose the viability. To address this issue, another kind of incorporation

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between cellulose and alginate was designed. Cellulose microgels were firstly

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encapsulated with L. plantarum cells in the same way, then coated with alginate and

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denoted as cores-shell gels. There was few L. plantarum cell releasing from

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cores-shell gels in SGF (Figure S5 f).

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The digital photograph of cores-shell gels was presented in Figure 4a. Cellulose

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microgels were the white spots in the microgels, and by contrast, alginate shell looked

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a little transparent. Figure 4b, c and d directly demonstrated the cores-shell structure

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of the microgels. Besides, the majority of blue fluorescence was shown in the core

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instead of shell, indicating that the distribution of L. plantarum cells mainly focused

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on the porous cellulose microgels rather than the alginate shell. The survival and

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release behaviors of L. plantarum cells from Ca-alginate gels and cores-shell gels in

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SIF were illustrated in Figure 5. The pH value of the gastric fluid in the human body

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is in the range of 1.8−5.0 along with the change of food intakes and human body, and

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pH 2 was chosen as the model gastric fluid to investigate the protection characteristics

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of L. plantarum cells. After exposure to SGF for 2 h, the release amount of viable L.

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plantarum cells from Ca-alginate gels was 9.7 ×103 cfu/mL in 10 min, and it kept

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rising up to 8.3 ×105 cfu/mL at 30 min, then the release rate decreased and the L.

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plantarum cells reached the maximum release degree of 4.2 ×106 cfu/mL at 270 min,

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but afterwards, the released amount of viable cells decreased to 1.1 ×106 cfu/mL at

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360 min. While in contrast, cores-shell gels showed more durable release of viable L.

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plantarum cells, 5.2 ×104 cfu/mL at 10 min, 3.4 ×105 cfu/mL at 30 min, then keeping


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rising up to 3.9 ×106 cfu/mL at 360 min. After exposure to SGF, Ca-alginate gels

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showed damaged viability in SIF after 270 min, and by contrast, the encapsulated

304

cells in cores-shell gels kept slow release without damage of viability, which

305

manifested cellulose microgels helped to sustaining the viable release for longer

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duration in SIF and affording better shelter for L. plantarum cells due to the porous

307

structure and rigid supporting property. The result of confocal laser scanning

308

microscopy already manifested probiotic cells penetrated into the core of microgels

309

(Figure 2), so L. plantarum cells were released in a sustainable state, the outermost

310

cells in microgels and few cells in alginate shell were released at first thus emerging

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the initial burst release, then followed by the release of inside L. plantarum cells with

312

decreased release rate. As for the release behavior in SIF without transiting through

313

SGF, it presented the best viable release behavior of L. plantarum cells without

314

damage from gastric acid, to some extent, it reflected the pristine loading amount of

315

bacteria in Ca-alginate gels and cores-shell gels. Without previous immersion in SGF,

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Ca-alginate gels showed an initial burst release of viable cells up to 2.04 ×105 cfu/mL

317

in 10 min, and then the release amount kept slowly rising until reaching the maximum

318

release degree of 7.2×106 cfu/mL at 180 min. The release file of cores-shell gels also

319

presented a burst release of viable cells up to 5.3×105 cfu/mL in 10 min, and kept

320

releasing up to 1.1×107 cfu/mL at 360 min. It should be noted that the maximum

321

viable release count of L. plantarum cells from cores-shell gels reached 1.1×107

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cfu/mL, indicating the loading capacity was at least higher than 1.1×108 cfu/g due to

323

the ten-fold dilution effect in SIF. This part of the work also indicated the bacteria



324

were most likely to be deposited at which regions of the gut. In conclusion, the

325

cores-shell gels performed more controlled release of L. plantarum cells lasting for

326

360 min, which implied that the entrapped L. plantarum cells could be liberated at

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large intestine regions where most of probiotics and a large concentration of

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indigenous bacterial species resided. In addition, there existed little difference

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between release profiles of Ca-alginate gels with and without exposure to SGF,

330

manifesting pH-responsible alginate ingredient did play a crucial role in protecting the

331

encapsulated cells. In the case of cores-shell gels, after exposure to SGF (for 2 hrs)

332

the amount of bacteria released in SIF decreased in compare to no exposure to SGF.

333

The main reasons were speculated as follows: to begin with the shelter effect of

334

porous cellulose microgels, cellulose was unable to be dissolved or digested, so

335

probiotic cells in cellulose microgels were not released rushly but in a sustainable and

336

more controlled way, which was good since it helped to achieve longer duration in

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intestine tracts so as to reach the desirable region. Secondly, the gel property of

338

alginate shell had changed to be more protective after immersion in SGF for 2h. As to

339

Ca-alginate gels, after exposure to SGF the amount of released cells at 10 mins

340

decreased in compare to no exposure to SGF, later then nearly increased to that of no

341

exposure to SGF.

342

In summary, modified porous cellulose microgels were pH-responsible scaffolds to

343

encapsulate probiotic with enhanced acid tolerant and high loading capacity. Cellulose

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was of great importance to improving the thermal stability and maintaining porous

345

and regular spherical structure of composite cellulose-alginate microgels. The


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probiotic cells tend to adhere heavily to the surface but not penetrate into the core of

347

CAM-4, while bacteria indeed reached the core of CAM-1 due to the more porous

348

structure. The composite microgels exhibited excellent pH-responsibility and also

349

remarkable loading capacity up to 109 cfu/g, but in SGF, the probiotic cells released

350

from the scaffold lost the viability. Therefore, cores-shell gels were fabricated to

351

improve the acid tolerance. As for cores-shell gels, L. plantarum cells were mainly

352

distributed in the cores section. Besides, the survival and release assays in SGF and

353

SIF showed few bacteria releasing from the cores-shell gels in SGF. When compared

354

with Ca-alginate gels, cores-shell gels showed more controlled release of L.

355

plantarum cells in SIF lasting for 360 min without damage of viability, implying that

356

the probiotic cells in cores-shell gels would reach the desire region in large intestine.

357

In cores-shell gels, cellulose microgels helped to sustaining the viable release for

358

longer duration in SIF and affording better shelter for L. plantarum cells due to the

359

porous structure and rigid supporting property, and also, it indicated a large loading

360

amount higher than 1.1 ×108 cfu/g in cores-shell gels. The fascinating cellulose

361

polysaccharide based scaffold is also promising for developing formulation with

362

prolonged stability and viability in biopharmaceutical industry.

363

Acknowledgment

364

This work was supported by the National Natural Science Foundation of China (No.

365

51273085, 51303142), and the National Science-technology Support Plan Projects

366

(No.2015BAD16B06), and project (2014PY024, 2662015PY099) by the Fundamental

367

Research Funds for the Central Universities.



368

Supporting information

369

Figure S1. FTIR spectra of CM, alginate sodium (ALG), and the composite

370

microgels.

371

Figure S2. XRD patterns of CM, ALG, and the composite microgels.

372

Figure S3. Thermal analysis of CM, ALG, and the composite microgels.

373

Figure S4. The images and size change of (a) CM, (b) CAM-1, (c) CAM-2, (d)

374

CAM-3 and (e) CAM-4 in different pH. The immersion of microgel in each buffer

375

lasted for 2 h.

376

Figure S5. The images of release probiotic cells from (a) CMs, (b) CAM-1, (c)

377

CAM-2, (d) CAM-3, (e) CAM-4 and (f) core-shell gels in SGF after staining with

378

methylene blue. Scale bar = 10 um.

379

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482 483 484



Figures:

Figure 1. SEM images and the surface morphology of microgels, a, b, c, d, e were CM, CAM-1, CAM-2, CAM-3 and CAM-4, respectively; and the surface morphology of the composite microgels (f, g, h, i, were CAM-1, CAM-2, CAM-3 and CAM-4, respectively) after encapsulation with L. plantarum cells.


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Figure 2. Laser scanning photograghs of CAM-1 (a, a’, a’’) and CAM-4 (b, b’, b’’) at scale of 1250 × 1250 um, a’’ represents the merged image of (a, a’) and b’’ represents the merged image of (b,b’).



Release count (CFU/mL)

1.0x108 8.0x107 6.0x107 4.0x107 2.0x107

CM CAM-1 CAM-2 CAM-3 CAM-4

5x100

0

60

120

180

240

300

360

Time (min) Figure 3. Release profile of probiotic cells from CMs, CAMs in simulated intestinal fluid for 6 h. 485


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Figure 4. The digital picture (a) and cross-sectional fluorescence image (b, c, d) of typical cores-shell gels encapsulated with L. plantarum cells. Scale bar = 100 um.

486



Page 28 of 29

107 107

106

10

5

104

a b

103 0

60

120

180

240

300



A

360

B

106

105

a b 104 0

60

120

Time (min)

180

240

300

360

Time (min)

Figure 5. Release files of viable L. plantarum cells from (A) Ca-alginate gels and (B) cores-shell gels in SIF: (a) without previous immersion of cores-shell gels in SGF and (b) with previous immersion in SGF for 2 h before transferring into SIF.


Page 29 of 29


47x26mm (600 x 600 DPI)


Porous Cellulose Microgel Particle: A Fascinating Host for the

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