Synbiotic encapsulation of probiotic Latobacillus plantarum by alginate -arabinoxylan composite microspheres

Accepted Manuscript Synbiotic encapsulation of probiotic Latobacillus plantarum by alginate -arabinoxylan composite microspheres Yue Wu, Genyi Zhang PII:

S0023-6438(18)30249-4

DOI:

10.1016/j.lwt.2018.03.034

Reference:

YFSTL 6963

To appear in:

LWT - Food Science and Technology

Received Date: 1 November 2017 Revised Date:

10 March 2018

Accepted Date: 13 March 2018

Please cite this article as: Wu, Y., Zhang, G., Synbiotic encapsulation of probiotic Latobacillus plantarum by alginate -arabinoxylan composite microspheres, LWT - Food Science and Technology (2018), doi: 10.1016/j.lwt.2018.03.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Synbiotic Encapsulation of Probiotic Latobacillus Plantarum by Alginate -

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Arabinoxylan Composite Microspheres

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Abbreviations:

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SA: sodium alginate

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AX: arabinoxylan

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AXOS: arabinoxylan oligosaccharides

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WUAX: water insoluble arabinoxylan

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FA: ferulic acid

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Yue Wu, and Genyi Zhang*

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State Key Laboratory of Food Science and Technology School of Food Science and Technology, Jiangnan University 1800, Lihu Road Wuxi, 214122 Jiangsu Province, PRC * Correspondence Author: [email protected] Telephone: 86-0510-85328726

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The probiotic Latobacillus

plantarum was encapsulated in microspheres prepared

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Abstract

through co-gelation of alginate (SA) and prebiotic arabinoxylan materials (AX), and the stability

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and survival rate of the probiotic in simulated gastrointestinal conditions were studied. The

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experimental results showed that the SA and AX were physically entangled to each other in the

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microspheres in which the AX molecules played a dominant role in probiotic encapsulation.

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Under simulated small intestinal conditions, a slower release rate of Latobacillus plantarum was

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observed when water-insoluble AX with the highest content of ferulic acid (1.78 µg/g AX) was

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used as the AX component in the microspheres. Compared to microspheres formed by alginate

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alone, the AX-SA composite microspheres significantly improved the encapsulation efficiency

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(2.5 folds), gastric stability (survival rate from 51.1% to 74.0%) and the bile salt resistance

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(survival rate from 70.6% to 81.6%). The incorporation of prebiotic arabinoxylan

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oligosaccharides (AXOS) into the AX-SA microspheres further significantly enhanced the

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encapsulation efficiency, gastric stability, bile salt resistance, as well as the storage stability.

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Thus, the co-formed AX-SA-AXOS synbiotic microsphere might be an ideal carrier for target

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delivery of probiotics.

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Key words: microencapsulation, prebiotics, probiotics Latobacillus plantarum, arabinoxylan,

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arabinoxylan oligosaccharide (AXOS)

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1. Introduction

The human gut harbors trillions of microbes (Whitman et al. 1998) (collectively termed

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as microbiota or microbiome) that play a crucial role to human health through their functions in

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regulating a variety of physiological processes including energy and fat metabolism, host

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immune system (both innate and adaptive) development, and barrier function to prevent

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pathogen invasion and colonization (Tremaroli and Backhed 2012). It is now well-recognized

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that many chronic diseases such as obesity (Verdam et al. 2013), type-2 diabetes (Larsen et al.

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2010) and inflammatory bowel disease (IBD) (Sartor and Mazmanian 2012) are associated with

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the dysbiosis of microbiota (Magrone and Jirillo 2013). Thus, microbiota has become a

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therapeutic target of IBD (Xavier 2016) and other metabolic diseases (Baars et al. 2015), and

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approaches to restore the host-microbial homeostasis are of great importance to the treatment of

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chronic diseases for improved health.

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Probiotics are ‘live microorganisms that, when administered in adequate amounts, confer

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a health benefit on the host’ (Sanders 2008). Intestinal strains of Lactobacillus, Bifidobacterium,

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and Enterococcus species are common examples of probiotics that are able to improve intestinal

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health, ameliorate lactose intolerance, and to reduce the risk of various diseases (Kechagia et al.

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2013). Mechanistically, the probiotics can modulate the transcriptional responses of the gut

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microbiota leading to discrete functional effects on the microbiota and host (Eloe-Fadrosh et al.

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2015). Another study also showed translational changes of enzymes related to many metabolic

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pathways by a consortium of fermented milk strains (McNulty et al. 2011). Related to probiotics,

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prebiotics are non-digestible carbohydrate materials that selectively promote the growth of

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probiotics, and prebiotics have been shown to have the potential to alter the composition of gut

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microbiota in a positive way (Parnell and Reimer 2012, Parnell and Reimer 2012, Yasmin et al.

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2015). Thus, synbiotics by combining probiotics and prebiotics might be an efficient measure to

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prevent or intervene the chronic diseases through regulating the host-microbial homeostasis. The successful delivery of probiotics to target location (colon) is essential for the

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probiotic microorganisms to exert their health functions to the host. However, the hostile

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gastrointestinal conditions may substantially reduce the number of transient or colonized

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probiotics. Therefore, new approaches which could protect and deliver probiotics are required,

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for instance, microencapsulation. Latobacillus plantarum, as a well-known probiotic with a

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variety of health benefits including treatment and prevention of enteric infections, post-antibiotic

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syndromes, inflammatory bowel disease (IBD) and colorectal cancer (Molin 2001), was used as

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the representative of probiotics in the current investigation.

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Alginate is a well-known biopolymer to encapsulate living microorganisms. However,

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Ca2+-induced alginate microcapsules are not stable under alkaline conditions, and co-gelation of

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alginate with other polymers such as chitosan (Chavarri et al. 2010), gelatinized starch (Khosravi

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Zanjani et al. 2014) and gelatin (Smitha 2017) was often used in practical applications.

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Arabinoxylan, as the cell wall component of cereal grains, consists of a linear β-(1-4) linked

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xylan backbone to which different side chains are attached, and importantly, the attached ferulic

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acids can lead to cross-linking between arabinoxylan molecules to form a gel matrix.

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Arabinoxylan is also a well-studied dietary fiber, and its fermentation not only produced health-

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promoting metabolites (Van den Abbeele et al. 2011) but also revealed its prebiotic property

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(Neyrinck et al. 2011). Comparatively, arabinoxylan-derived arabinoxylan oligosaccharides

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(AXOS) have been shown to be more efficient to stimulate the growth of beneficial intestinal

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microbiota and to produce functional short chain fatty acids (Broekaert et al. 2011). However,

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little information is available on the arabinoxylan and/or AXOS, as a prebiotic, in probiotic

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encapsulation. So a synbiotic microencapsulation of probiotic Latobacillus plantarum by an

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alginate-arabinoxylan/AXOS composite microspheres was studied, which could help to gain

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insight into the impact of arabinoxylan and/or AXOS on the activity of encapsulated probiotics

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in simulated gastrointestinal conditions.

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2. Materials and Methods

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2.1 Materials

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The enzymes of laccase (EC 1.10.3.2, p-diphenol:dioxygen oxidoreductases), heat-stable

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α-amylase, and β-1,4 xylanase were purchased from Sigma Aldrich (Shanghai, China). Sodium

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alginate and pepsin were from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Maize

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bran was purchased from a local store.

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2.2 Extraction and analysis of maize arabinoxylan

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The arabinoxylan was extracted from maize bran according to a literature method

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(Bataillon et al. 1998). Briefly, the maize bran was pretreated with heat-stable α-amylase to

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remove the starch, and then pepsin to remove the protein. The lignin in the bran was liberated

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with 40% sodium chloride at 70°C for 2 h. The water-soluble arabinoxylan in the supernatant

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was precipitated by ethanol (65%) after centrifugation at 10,000×g for 10 min. The water-

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insoluble arabinoxylan was obtained after the precipitated bran sample was incubated in a

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solution of NaOH (30%) at 80°C. The arabinoxylan oligosaccharides (AXOS) was prepared by

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an enzymatic method (Pollet et al. 2012) using B. subtilis β-1,4 xylanase at 50°C for different

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time periods (1, 2 and 4 h). The molecular weight of arabinoxylan and AXOS was measured by a

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HPLC method (Zhang et al. 2003) using pullulan molecular weight standard (Megazyme

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International Ireland). The monosaccharide composition of the extracted arabinoxylan was

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analyzed according to a literature report (Mazumder and York 2010) using HPLC after acid

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hydrolysis of the samples. The gelation properties of arabinoxylan (2.0%,w/v) were measured

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using an AR-G2 rheometer (TA Instruments-Waters LLC, Shanghai, China), and the changes of

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storage modulus (Pa) along the gelation time (right after the addition of laccase (1.67 nkat/mg

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AX at 25°C)) were recorded to reflect the gelation property of arabinoxylan.

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2.3 Ferulic acid content measurement

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The content of ferulic acid was quantified by using a HPLC method (Kareparamban et al.

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2013). Specifically, arabinoxylan (50 mg) was dissolved in 5 mL of purified water, and

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arabinoxylan solution (1.0 mL) was mixed with 2.0 mol / L NaOH solution (1.0 mL) for 2 h in a

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dark room. The pH was adjusted to 2.0 with HCl (1.0 mol/L), and then ether (5 mL) was used to

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extract the ferulic acid. After the ether extract was evaporated at 30 °C, 1.0 mL of methanol was

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used to dissolve the extract and then the internal standard (trans-cinnamic acid) was added. After

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the final extract was filtered with a filter film (pore size = 0.45 µm), HPLC was used to measure

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the content of ferulic acid. Chromatographic conditions: column C18 (particle size 5 µm, 4.6 x

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150 mm id); gradient eluent: methanol and 0.05% H3PO4 solution; elution temperature 35°C ;

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detection wavelength 320 nm; elution rate of 1.0 mL per min; elution gradient: linear gradient

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from 25:75 to 90:10 in 0-18 min, from 90:10 to 25:75 in 18-19 min, and 25:75 in 19-24 min. The

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area under the ferulic acid peak was compared to the internal standard to calculate its content.

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2.4 Alginate-arabinoxylan composite microsphere preparation and FTIR analysis

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Alginate solution (1.8%, w/v) with different amount of arabinoxylan (AX) was mixed.

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Then, the laccase (1.67 nkat/mg AX ) was added and mixed at 25 °C for 10 min. The mixture

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was then dropped into a CaCl2 solution (1.0%) through a syringe needle (length 3/8, gauge 20).

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After gelation for 1.0 h, the formed microspheres were filtered with purified water 2 times, then

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freeze-dried before subjected to FITR analysis. The dried microspheres were crushed into

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powder and subjected to infrared spectroscopy in the range of 4000 to 400 cm-1 after mixing with

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KBr (KBr = 1: 100).

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2.5 Probiotic culture and the fermentation of arabinoxylan MRS liquid medium was used to culture the frozen bacteria at 37 °C for passages until a

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complete activation. The activated Lactobacillus plantarum was then inoculated into carbon

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source-free culture medium (containing arabinoxylan or AXOS (1.0% w/v)) at 37 °C and

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allowed to stand for 48 h. A negative control (no carbon medium) and a positive control (1.0%

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glucose) were used, and the number of viable bacteria and the pH of the culture medium were

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measured to reflect the fermentability of arabinoxylan with different molecular weights.

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2.6 Microencapsulation of probiotics

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The microencapsulation was performed based on the method reported by Cheow et al.

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(2014) with minor modifications. Briefly, the bacterial culture (after centrifugation of log phase

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bacteria) was added into the sterilized alginate-arabinoxylan (or the mixed solution containing

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AXOS 0.5%) solution at a ratio of 1:10. Then the laccase (1.67 nkat / mg AX), after passing

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through filter film (0.22 µm), was added into the mixed solution at 4 °C. After mixing for 10

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min, the mixed solution was dropped into the bacteria-free calcium chloride solution (1.0%)

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through a digital gear drive pump (Cole-parmer, WX-75211-30, at a speed of 50 rpm). After 30-

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60 min in the CaCl2 solution, the microsphere was filtered and washed with sterile water 2 times.

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Then, the freshly prepared microspheres were mixed with 15% glycerol, and stored in liquid

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nitrogen. Freeze-dried microspheres were used for long-term storage analysis.

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2.7 Survival rate of probiotics in simulated gastrointestinal conditions

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2.7.1 Encapsulation efficiency

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Microspheres (1.0 g) were added into 10 mL phosphate buffer solution (pH 7.4) and

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mixed on a shaker at 100 rpm for 10 min. An Ultra-turax T18 homogenizer was then used to

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homogenize the microspheres for 1.0 min, and MRS agar plate assay was employed to count the

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total cell numbers after 48 h incubation at 37 °C. The encapsulation efficiency EY = N / N0 ×

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100%, where N referred to the total number of viable cells encapsulated; N0 referred to the total

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number of live cells in the initial concentrated bacteria culture.

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2.7.2 Susceptibility to gastric and bile salt conditions

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The susceptibility of the encapsulated probiotics to gastric condition was tested according

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to a reported method (Mandal et al. 2014). Specifically, microspheres（1.0 g）and sterilized

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artificial gastric juice (3% pepsin, 2% NaCl and pH 2.0) (the pepsin solution was passed through

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a filter with pore size of 0.22µm) were incubated in a water bath (37 °C) shaker at 100 rpm.

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After incubation for 60 and 120 min, samples were collected and the viable cells were counted as

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described above. Similar to the gastric condition, the resistance to high bile salt was also tested in

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the same steps except the artificial gastric juice was replaced by sterilized high bile salt solution

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(bile salt 2 g / L, 100 mmol / L NaOH , pH 6.8) and an incubation time of 4 h. The control

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experiment was carried out with 1.0 mL Lactobacillus plantarum. The survival rate was

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calculated as follows:

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Survival rate X = (lgN / lgN0) x 100%

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Where N0 was the number of viable cells of the original bacteria; N was the number of viable

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cells treated by artificial gastric juice or high bile salt solution.

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2.7.3 Release rate of the probiotics in simulated intestinal conditions

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The release rate was measured according to the following steps: microcapsules (1.0 g)

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was mixed with 9.0 mL artificial small intestinal fluid (1% trypsin, pH 6.8) and incubated at 8

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37°C water bath shaker (100 rpm). Then, 0.1 mL solution at 0, 1, 2, 3, 4, 5 and 6 h was taken out

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to count the viable cells based on the above method. In the meantime, 0.1 mL artificial small

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intestinal fluid was added back to the tubes to maintain the volume. The artificial fluid could also

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be small intestinal fluid containing β-1,4-xylanase (1.0 g/L) in a PBS solution (pH = 7.4). The

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storage stability was also tested after the freeze-dried microcapsules were stored in sterile and

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sealed vials at room temperature for different days.

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2.8 Statistical analysis

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The experiments were carried out in triplicates, and software package of SPSS 19.0 was

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used for One-way ANOVA analysis. A level of p < 0.05 was considered statistical significant

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different. The experimental results were expressed as mean ± standard deviation.

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3. Results

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3.1 The gelation of arabinoxylan

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The ferulic acid, as the main phenolic compound covalently attached to the arabinoxylan,

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is the basis to arabinoxylan (AX) gelation. In the current investigation, the content of ferulic acid

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was 2.23 µg/gAX and 1.78 µg/gAX for water-soluble arabinoxylan (A/X = 0.63) and water-

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insoluble counterpart (A/X = 0.56), respectively. Additionally, the ratio of A/X suggested that

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more side chains (higher ratio) were present in the water-soluble AX than those in water-

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insoluble AX. But the molecular weight of water-soluble arabinoxylan (1.0×105) was lower than

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water-insoluble arabinoxylan (2.5×105). Although both the ferulic acid content and molecular

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structure of AX contribute to the gelation property of AX, the rheological study of the

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arabinoxylan gelation in the presence of laccase showed a higher storage modulus for water

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insoluble arabinoxylan (lower A/X) than the water-soluble counterpart (Fig 1A), implying water-

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insoluble AX can form a rigid gel matrix at lower concentrations. Thus, water-insoluble

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arabinoxylan was chosen as the material for the following studies. The gelation process was also

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evidenced by a substantial decrease of monomeric ferulic acid and a concomitant increase of

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dimeric and trimeric ferulic acids along the time of gelation of water-insoluble AX (Fig 1B).

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Apparently, 30-40 min was enough for the complete gelation of arabinoxylan under the used

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conditions.

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3.2 The impact of ferulic acid content and the concentration of arabinoxylan on the stability

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and release property of encapsulated probiotics

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Since ferulic acid is the basis of arabinoxylan gelation, the impact of the content of

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ferulic acid attached to arabinoxylan on the Lactobacillus plantarum encapsulation efficiency

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was examined. As shown in Table 1, after the probiotic was encapsulated in the alginate-

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arabinoxylan (SA-AX) composite microspheres (sodium alginate: 1.8%, CaCl2: 1.0%, 30 min),

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the water-insoluble arabinoxylan with the highest content of ferulic acid (1.78 µg/gAX) had the

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highest encapsulation efficiency and survival rate in gastric conditions. But the release of the

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encapsulated probiotics was the lowest (Fig 2). Therefore, the tight matrix formed through

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ferulic acid-mediated cross-linking of arabinoxylan molecules might be the main reason for the

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observed results.

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The impact of arabinoxylan content in the composite microsphere on the encapsulation

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efficiency were also examined. As shown in Table 2, the highest encapsulation efficiency was

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observed in the group with 2% arabinoxylan while the highest survival rate in the gastric

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condition was noted in the group with 6% arabinoxylan. For the release rate of probiotics in the

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mimicked small intestinal condition (Fig 3), the lowest release rate was found in the group with

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the highest content of arabinoxylan (8%). However, when the overall retention rate of probiotics

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after treatment by simulated gastrointestinal condition was considered, the group with 4% (w/v)

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arabinoxylan was the ideal composite microspheres with a relatively higher encapsulation

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efficiency and survival rate as well. Thus, a concentration of 4% arabinoxylan was chosen for the

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preparation of the SA-AX composite microspheres to encapsulate the probiotic Lactobacillus

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plantarum.

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3.3 Encapsulation of probiotic Lactobacillus plantarum

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The encapsulation of the probiotics involved a proper combination of many parameters to

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achieve the highest retention rate of viable Lactobacillus plantarum after treatment by simulated

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gastrointestinal conditions. The experimental results showed that 1.8% SA, 4% AX, 1.67 nkat /

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mg AX, 1.0% CaCl2 with a gelation time of 30-40 min could yield desired microspheres to

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encapsulate the probiotics. Compared to microspheres prepared with alginate (SA) alone, the

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SA-AX composite microsphere also substantially improved the survival rate of the Lactobacillus

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plantarum after bile-salt treatment (Table 3).

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Further analysis of the microspheres by FTIR showed that there was no covalent

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connection between alginate and arabinoxylan (Supplemental Fig S1) indicating their physical

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entanglement in the microspheres. The physically entangled alginate and arabinoxylan molecules

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and their co-gelation may together entrap the bacteria in the SA-AX network, which was

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evidenced by an almost complete release of bacteria when the microsphere was treated by β-1, 4-

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xylanase (Fig 4) that cleaved the arabinoxylan molecules. The degradation of arabinoxylan by β-

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1, 4-xylanase might lead to a disruption of the physical structure of the microsphere and a

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complete release of bacteria. Thus, arabinoxylan played a dominant role in the microsphere to

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encapsulate the probiotic Lactobacillus plantarum, which was also supported by the above

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results (Table 1, Fig2).

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3.3 The in vitro fermentation of arabinoxylan by probiotic Lactobacillus plantarum

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It is known that prebiotics are carbohydrate materials (such as oligo-fructose) that

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selectively promote the proliferation of probiotics. To test the prebiotic property of arabinoxylan,

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an in vitro fermentation of arabinoxylan with different molecular weights was carried out. As

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shown in Fig 5, the pH of the culture media decreased significantly along the reduction of

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molecular weight of arabinoxylan and arabinozylan oligosaccharides (AXOS) while the number

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of live Lactobacillus plantarum was concomitantly increasing, confirming the prebiotic property

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of arabinoxylan, particularly AXOS.

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3.4 SA-AX-AXOS composite microsphere for the delivery of Lactobacillus plantarum

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The significant prebiotic property of AXOS indicates it could be incorporated into the

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delivery system as a synbiotic encapsulation of probiotics to facilitate its recovery, to maintain

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its viability, and to improve its stress-resistance during the delivery process. Thus, we also

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fabricated SA-AX-AXOS synbiotic microspheres and examined the impact of AXOS on the

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delivery efficiency.

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When 0.5% AXOS (Mw:4.5×104, 3.3×104, 2.1×104) was incorporated into the SA-AX

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microsphere under the same preparation condition, the encapsulation efficiency was improved

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significantly from 67.1% to 85.4% when the AXOS with the lowest molecular weight was used

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(Table 4), which suggests that the fermentable AXOS by Lactobacillus plantarum does influence

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the efficiency of the delivery system. Meanwhile, the gastric condition-resistance was also

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improved (Table 4) although the release rate under mimicked small intestinal condition was not

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affected (data not shown). Further analysis showed that 0.5% AXOS was likely the optimal

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content for encapsulation of Lactobacillus plantarum when the AXOS3 (Mw = 2.1×104 ) was

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used. Certainly, highest encapsulation efficiency and gastric condition-resistance were observed

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when 0.5% AXOS3 was incorporated into the synbiotic microspheres (Table 5). Consistently,

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the incorporation of AXOS under the optimal condition also significantly improved the storage

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stability of the microspheres maintained at room temperature (Fig 6).

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4. Discussion With the increased awareness of the health benefits of probiotic microorganisms,

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probiotic encapsulation is often used to improve the stability of probiotic microorganism (Anal

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and Singh 2007) and their survival rate during transit through the hostile condition of

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gastrointestinal tract (Shori 2017). Alginate is a well studied encapsulated polymer, which is

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easy to form gel matrix to entrap probiotics due to its ionic property. There have been many

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reports on probiotic encapsulation using alginate alone (Li et al. 2017) or in combination with

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other polymers such as chitosan and psyllium (Lotfipour et al. 2012, Yeung et al. 2016),

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demonstrating the long-lasting interest in alginate as the encapsulation agent.

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Arabinoxylan is a cereal-sourced dietary fiber, and it is highly fermented by the

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microbiota to result in multiple health benefits (Saeed et al. 2011), and a literature report also

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showed its potential prebiotic property (Broekaert et al. 2011). Although there was a report

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using arabinoxylan to encapsulate probiotics (Morales-Ortega et al. 2014), little information is

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available concerning its co-encapsulation with other polymers. In the current study, significant

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improvement in encapsulation efficiency and gastric stability of the probiotic Lactobacillus

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plantarum was observed in the SA-AX composite microspheres. Thus, the incorporation of

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arabinoxylan into the microspheres substantially improves the structural stability of the capsules

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and the acid-resistance of encapsulated probiotics. The dominant role of arabinoxylan in the

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probiotic encapsulation, as suggested from the experimental results (Fig. 4, Table 1&2), is

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consistent with the improved quality of probiotic encapsulation. Additionally, the high storage

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modulus of water-insoluble arabinoxylan gelation (Fig 1) implies arabinoxylan with a high

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molecular weight may have a long-range cross-linking between arabinoxylan molecules, which

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might provide more space to improve the encapsulation efficiency. In the meantime, the

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interactions between arabinoxylan and alginate during gelation may synergistically improve the

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physical stability of the microspheres leading to a high resistance to acid and bile salt.

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Apparently, high encapsulation efficiency (porous structure) and high acid-resistance (rigid

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structure) are contradictory to each other, and that is why a proper combination of alginate and

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arabinoxylan is required to achieve a high quality encapsulation. Certainly, as the microsphere

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was produced through the gelation of alginate in the presence of Ca2+ and ferulic acid-mediated

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cross-linking of arabinoxylan, the physiochemical property of the microspheres can be fine-tuned

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to achieve desired encapsulation outcome for practical applications.

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The prebiotic property of arabinoxylan, particularly arabinoxylan-derived oligosaccharide

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(AXOS), has been demonstrated in many literature studies (Damen et al. 2011, Neyrinck et al.

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2011, Hald et al. 2016) and in our current study. Similar to the protective effect of the prebiotic

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galactooligosaccharide on the alginate-chitosan encapsulated probiotics (Krasaekoopt and

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Watcharapoka 2014) and the efficient alginate-inulin synbiotic encapsulation to deliver the

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probiotics to the colon (Atia et al. 2017), the prebiotic AXOS in the microcapsules significantly

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improved the encapsulation efficiency of Lactobacillus plantarum and its storage stability. The

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incorporation of AXOS may influence the structure of the SA-AX composite microspheres

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leading to an increased encapsulation efficiency, and the AXOS may also protect the capsulated

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probiotics from low-pH exposure. Therefore, AX-SA-AXOS synbiotic composite microspheres

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could efficiently improve the stability and survival rate of probiotics in simulated gastrointestinal

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conditions, and can be used as an ideal carrier to deliver the probiotic to target location for its

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survival and colonization.

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5. Conclusion Microencapsulation to improve the stability of encapsulated probiotics has been an active

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research area for decades due to its wide industrial interest. The increasing awareness of the

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health benefits of probiotics further promotes the interest of consumers. An arabinoxylan-

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alginate-AXOS synbiotic microspheres used to encapsulate the prebiotic Lactobacillus

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plantarum combined both probiotics and prebiotics in the delivery system. Positive results of

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high encapsulation efficiency and high resistance to gastrointestinal conditions make the

347

synbiotic encapsulation an efficient approach to deliver probiotics, which also warrants further in

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vivo study to explore the colonization of probiotics and resulted health benefits.

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Declaration of interest

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The author declare no conflict of interest.

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Acknowledgement

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This work was supported by the National Natural Science Foundation of China (No.31471585).

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Reference: 1. Anal, A. and H. Singh (2007). Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends in Food Science & Technology 18, 240251. 2. Atia, A., A. I. Gomma, I. Fliss, E. Beyssac, G. Garrait and M. Subirade (2017). Molecular and biopharmaceutical investigation of alginate-inulin synbiotic coencapsulation of probiotic to target the colon. Journal of Microencapsul 34(2): 171-184. 3. Baars, A., A. Oosting, J. Knol, J. Garssen and J. van Bergenhenegouwen (2015). The gut microbiota as a therapeutic target in IBD and metabolic disease: A role for the bile acid receptors FXR and TGR5. Microorganisms 3(4): 641-666. 4. Bataillon, M., P. Mathaly, A. P. Nunes Cardinali and F. Duchiron (1998). Extraction and purification of arabinoxylan from destarched wheat bran in a pilot scale. Industrial Crops and Products 8(1): 37-43. 5. Broekaert, W. F., C. M. Courtin, K. Verbeke, T. Van de Wiele, W. Verstraete and J. A. Delcour (2011). Prebiotic and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides, and xylooligosaccharides. Critical Review in Food Science and Nutrition 51(2): 178-194. 6. Broekaert, W. F., C. M. Courtin, K. Verbeke, T. Van de Wiele, W. Verstraete and J. A. Delcour (2011). Prebiotic and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides, and xylooligosaccharides. Critical Reviews in Food Science and Nutrition 51(2): 178-194. 7. Chavarri, M., I. Maranon, R. Ares, F. C. Ibanez, F. Marzo and C. Villaran Mdel (2010). Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal conditions. Int J Food Microbiol 142(1-2): 185-189. 8. Cheow, W. S., T. Y. Kiew and K. Hadinoto (2014). Controlled release of Lactobacillus rhamnosus biofilm probiotics from alginate-locust bean gum microcapsules. Carbohydr ate Polymers 103: 587-595. 9. Damen, B., J. Verspreet, A. Pollet, W. F. Broekaert, J. A. Delcour and C. M. Courtin (2011). Prebiotic effects and intestinal fermentation of cereal arabinoxylans and arabinoxylan oligosaccharides in rats depend strongly on their structural properties and joint presence. Molecular Nutrition and Food Research 55(12): 1862-1874. 10. Eloe-Fadrosh, E. A., A. Brady, J. Crabtree, E. F. Drabek, B. Ma, A. Mahurkar, J. Ravel, M. Haverkamp, A.-M. Fiorino, C. Botelho, I. Andreyeva, P. L. Hibberd and C. M. Fraser (2015). Functional dynamics of the gut microbiome in elderly people during probiotic consumption. mBio 6(2). 11. Hald, S., A. G. Schioldan, M. E. Moore, A. Dige, H. N. Lærke, J. Agnholt, K. E. Bach Knudsen, K. Hermansen, M. L. Marco, S. Gregersen and J. F. Dahlerup (2016). Effects of arabinoxylan and resistant starch on intestinal microbiota and short-chain fatty acids in subjects with metabolic syndrome: A randomised crossover study. PLoS ONE 11(7): e0159223. 12. Kareparamban, J. A., P. H. Nikam, A. P. Jadhav and V. J. Kadam (2013). A validated highperformance liquid chromatograhy method for estimation of ferulic acid in asafoetida and polyherbal preparation. Indian Journal of Pharmaceutical Sciences 75(4): 493-495.

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26. Parnell, J. A. and R. A. Reimer (2012). Prebiotic fiber modulation of the gut microbiota improves risk factors for obesity and the metabolic syndrome. Gut microbes 3(1): 10.4161/gmic.19246. 27. Parnell, J. A. and R. A. Reimer (2012). Prebiotic fibres dose-dependently increase satiety hormones and alter Bacteroidetes and Firmicutes in lean and obese JCR:LA-cp rats. British Journal of Nutrition 107(4): 601-613. 28. Pollet, A., V. Van Craeyveld, T. Van de Wiele, W. Verstraete, J. A. Delcour and C. M. Courtin (2012). In vitro fermentation of arabinoxylan oligosaccharides and low molecular mass arabinoxylans with different structural properties from wheat (Triticum aestivum L.) bran and psyllium (Plantago ovata Forsk) seed husk. Journal of Agricultural and Food Chemistry 60(4): 946-954. 29. Saeed, F., I. Pasha, F. M. Anjum and M. T. Sultan (2011). Arabinoxylans and arabinogalactans: a comprehensive treatise. Crit Rev Food Sci Nutr 51(5): 467-476. 30. Sanders, M. E. (2008). Probiotics: Definition, Sources, Selection, and Uses. Clinical Infectious Diseases 46(Supplement_2): S58-S61. 31. Sartor, R. B. and S. K. Mazmanian (2012). Intestinal Microbes in Inflammatory Bowel Diseases. The American Journal of Gastroenterology Suppl 1(1): 15-21. 32. Shori, A. B. (2017). Microencapsulation Improved Probiotics Survival During Gastric Transit. HAYATI Journal of Biosciences 24(1): 1-5. 33. Smitha, M. ( 2017). Microencapsulation of Probiotics by Calcium Alginate and Gelatin and Evaluation of its Survival in Simulated Human Gastro-Intestinal Condition. Internatioal Journal ofCurrent Microbiology and Applied Sciences 6(4 ): 2080-2087. 34. Tremaroli, V. and F. Backhed (2012). Functional interactions between the gut microbiota and host metabolism. Nature 489(7415): 242-249. 35. Van den Abbeele, P., P. Gérard, S. Rabot, A. Bruneau, S. El Aidy, M. Derrien, M. Kleerebezem, E. G. Zoetendal, H. Smidt, W. Verstraete, T. Van de Wiele and S. Possemiers (2011). Arabinoxylans and inulin differentially modulate the mucosal and luminal gut microbiota and mucin-degradation in humanized rats. Environmental Microbiology 13(10): 2667-2680. 36. Verdam, F. J., S. Fuentes, C. de Jonge, E. G. Zoetendal, R. Erbil, J. W. Greve, W. A. Buurman, W. M. de Vos and S. S. Rensen (2013). Human intestinal microbiota composition is associated with local and systemic inflammation in obesity. Obesity 21(12): E607-E615. 37. Whitman, W. B., D. C. Coleman and W. J. Wiebe (1998). Prokaryotes: the unseen majority. Proceedings of the National Academy of Sciences95(12): 6578-6583 38. Xavier, R. J. (2016). Microbiota as Therapeutic Targets. Digestive Diseases 34(5): 558-565. 39. Yasmin, A., M. S. Butt, M. Afzaal, M. van Baak, M. T. Nadeem and M. Z. Shahid (2015). Prebiotics, gut microbiota and metabolic risks: Unveiling the relationship. Journal of Functional Foods 17: 189-201. 40. Yeung, T. W., E. F. Üçok, K. A. Tiani, D. J. McClements and D. A. Sela (2016). Microencapsulation in Alginate and Chitosan Microgels to Enhance Viability of Bifidobacterium longum for Oral Delivery. Frontiers in Microbiology 7: 494. 41. Zhang, G., M. D. Maladen and B. R. Hamaker (2003). Detection of a novel three component complex consisting of starch, protein, and free fatty acids. Journal of Agricultural and Food Chemistry 51(9): 2801-2805.

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Figure captions:

496

Figure 1. The storage modulus (A) and changes of different forms of ferulic acid during laccase-

497

catalyzed gelation of water insoluble arabinoxylan (B). AG’: modulus of alkaline-extracted

498

arabinoxylan, WG’: modulus of water-soluble arabinoxylan. FA: ferulic acid, Di-FA: dimeric

499

ferulic acid, Tri-FA: trimeric ferulic acid.

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Figure 2. The impact of ferulic acid on the release of microencapsulated probiotic Lactobacillus

502

plantarum in simulated intestinal juice. SA: alginate, WUAX: water-insoluble arabinoxylan,

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ferulic acid content in WUAX (1.78 ± 0.01 µg/gAX), WUAX1 (1.17 ± 0.03 µg/gAX), and

504

WUAX2 (0.54 ± 0.05 µg/gAX).

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Figure 3. The influence of the concentration of water-insoluble arabinoxylan (WUAX) on the

507

release of probiotic Lactobacillus plantarum in simulated small intestinal juice.

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Figure 4. The release profile of Lactobacillus plantarum from the SA-AX composite

510

microsphere in the presence of different concentrations of β1,4-xylanase incubated at 37°C in

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phosphate buffer ( pH 6.8).

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Figure 5. The fermentation profiles of arabinoxylan and arabinoxylan oligosaccharides by

514

probiotic Lactobacillus plantarum. The control: no carbon source in the medium. Different

515

letters represent statistically significant difference at p<0.05.

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Figure 6. The storage stability of encapsulated Lactobacillus plantarum at room temperature for

518

different days. Different letters represent statistically significant difference at p<0.05.

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521 522 523 524 525 526 527 528 529

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Table 1 Effects of contents of ferulic acid on the survival of microencapsulated Lactobacillus plantarum in simulated gastric juice. Encapsulation

Viable cells (log(CFU/mL))

efficiency (%)

0h

1h

SA

21.61±3.72a

8.24±0.15

6.03±0.31

4.34±0.23

52.67±1.90a

WUAX-SA

67.23±1.15d

8.89±0.08

7.34±0.13

6.75±0.08

75.93±0.63d

WUAX1-SA

51.27±2.32c

8.84±0.12

7.02±0.21

6.12±0.04

69.22±0.95c

WUAX2-SA

44.62±1.70b

8.76±0.05

Survival rate

SC

2h

M AN U

Microsphere

6.53±0.25

5.42±0.15

（%）

61.99±1.95b

530

Note：Different superscript letters represent significant differences at p<0.05. SA: alginate alone,

531

WUAX: water insoluble arabinoxylan, Ferulic acid in WUAX: 1.78 µg/mg AX, Ferulic acid in WUAX1：

532

1.17 µg/mg AX, Ferulic acid in WUAX2：0.54 µg/mg AX.

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0

22.42±3.71b

8.08±0.06

2

79.64±1.12e

8.96±0.08

4

67.83±2.45d

8.75±0.17

6

57.91±2.12c

8.58±0.16

8

18.91±4.53a

8.01±0.12

SC

Table 2. Effects of the concentration of arabinoxylan on the survival of AS-AX microencapsulated Lactobacillus plantarum in simulated gastric juice. WUAX Encapsulation Viable cells (log(CFU/mL)) Survival rate conc.(%) enfficiency (%) 0h 1h 2h (%) Control N/A 8.77±0.07 5.36±0.24 ND N/A3.85±0.23

47.65±1.93a

7.04±0.23

6.31±0.13

70.42±1.85b

7.12±0.24

6.42±0.1

73.37±2.32c

7.31±0.25

6.58±0.12

76.69±4.37d

6.37±0.14

5.71±0.15

71.29±3.30c

5.76±0.26

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Note：ND- not detected, different superscript letters represent significant differences at p<0.05. WUAX:

542

water insoluble arabinoxylan.

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544 545 546

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Table 3. The survival rate of microencapsulated Lactobacillus plantarum in bile solution. Viable cells (log(CFU/mL)) 0h 2h

Survival rate

4h

（%）

69.7±5.46a

Control

8.79±0.36

7.01±0.31

6.25±0.20

SA

8.03±0.48

6.35±0.13

WUAX-SA

8.78±0.05

7.89±0.14

SC

WUAX（%）

5.73±0.31

70.67±4.32b

7.13±0.33

81.67±0.27c

Note: different superscript letters represent significant differences at p<0.05. WUAX-SA: water insoluble

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arabinoxylan and alginate composite microsphere.

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Table 4. Effects of AXOS with different molecular weights on the survival of microencapsulated Lactobacillus plantarum in simulated gastric juice. Encapsulation Viable cells（log(CFU/mL)) Survival Rate Microsphere

564 565

2h

SA

22.42±3.71a

8.39±0.09

5.64±0.31

4.34±0.24

51.74±2.29a

WUAX-SA

67.15±1.12b

8.87±0.13

7.07±0.26

6.41±0.22

72.31±3.51b

AXOS1

80.63±3.52c

8.89±0.18

7.39±0.19

6.73±0.16

75.70±0.36c

AXOS2

82.16±3.71c

8.93±0.18

7.63±0.17

6.89±0.19

77.47±4.06d

AXOS3

85.45±2.36d

8.91±0.18

7.73±0.17

7.05±0.19

79.03±0.85e

SC

1h

Note: different superscript letters represent significant differences at p<0.05. WUAX-SA: water insoluble arabinoxylan and alginate composite microspheres. AXOS: arabinoxylan oligosaccharides, AXOS(1-3) Mw:4.5×104, 3.3×104, 2.1×104.

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0h

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（%）

v

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561

efficiency (%)

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Table 5 Effects of the concentration AXOS on the survival of microencapsulated Lactobacillus plantarum in simulated gastric juice. Encapsulation AXOS Survival Viable cells（log(CFU/mL)) Conc.(%）

efficiency (%)

WUAX-SA

2h

69.64±2.82a

8.76±0.28

7.29±0.17

6.31±0.39

72.04±3.01a

0.1

80.16±3.25c

8.86±0.21

7.27±0.21

6.70±0.49

76.00±3.93b

0.5

86.13±3.72d

8.84±0.37

7.54±0.30

6.95±0.27

78.86±2.57c

576

72.45±2.61b

SC

1h

1.0 575

rate（%）

0h

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8.66±0.28

7.42±0.36

6.79±0.35

78.36±1.84c

Note: different superscripts represent statistical significance at p<0.05. AXOS: arabinoxylan oligosaccharides, WUAX-SA: water insoluble arabinoxylan and alginate composite microspheres.

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580 581 582 583 584 585 586 587 588 589 590

593

300

594 595 596 597

(A)

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Storage modulus (Pa)

592

SC

591

250 200 150 100

AG' WG'

50

598

0 0

500

1000

TE D

599

1500

2000

Time (sec)

600 601

(B)

603

606 607

1.2

0.8

AC C

604 605

FA Di-FA Tri-F A

EP

Ferulic acid content (µg/gAX)

602

1.6

0.4

0.0

608

0

100

150

200

250

Time (min)

609 610

50

Figure 1.

611 612 25

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613 614 615

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616 617 618 619

SC

620 621 622

626 627 628

636

2

3

4

5

6

EP

AC C

635

1

Time (h)

Figure 2.

634

5

0

630

633

6

4

629

632

7

TE D

Living cells (Logcfu/mL)

625

631

SA WUAX-SA WUAX1-SA WUAX2-SA

8

624

M AN U

9

623

637 638 639

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640 641 642 643

9

647 648 649

7

6

SC

646

8

5

4

650

0

651 652

1

M AN U

Released cells (Logcfu/mL))

645

RI PT

0% 2% 4% 6% 8%

644

2

3

4

5

6

Time (h)

Figure 3.

AC C

EP

TE D

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660 661 662 663 664

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0.1% 0.5% Contr ol

9

8

7

6

SC

658

5

4

665

0

1

M AN U

Released L. Plantarum (LogCFU/mL)

10

2

3

4

5

6

Time (h)

666 667

Figure 4.

AC C

EP

TE D

668

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669 670 671

674 7

a

B

pH LogCFU/mL

a

675

b

6

678 679

d 5 D

4 3 2 A

681 0

682

6

2

C

1

0

Control Glucose

AXOS1 AXOS2 AXOS3

EP

TE D

Figure 5.

AX

AC C

684

E

4

680

683

DE

SC

677

8

c

M AN U

Culture medium pH

676

10

Living cells (LogCFU/mL)

673

RI PT

672

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685 686 D ay 0 D ay 15 D ay 60

10

689 690 691 692 693 694

8

a b

c

a b

c

6

4

2

0

Figure 6.

SA-AX-AXOS

AC C

EP

TE D

697

SA-AX

M AN U

SA

695 696

b

RI PT

688

a

a

SC

Number of live cells (LogCFU/mL)

687

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Composite microsphere is produced by co-gelation of alginate and arabinoxylan The composite microsphere significantly improve the survival rate of probiotics The prebiotic arabinoxylan oligosaccharides enhance the encapsulation efficacy Synbiotic encapsulation can be used as an efficient carrier to deliver probiotics