Arabinoxylans, gut microbiota and immunity

Accepted Manuscript Title: Arabinoxylans, gut microbiota and immunity Author: Mihiri Mendis Estelle Leclerc Senay Simsek PII: DOI: Reference:

S0144-8617(15)01158-3 http://dx.doi.org/doi:10.1016/j.carbpol.2015.11.068 CARP 10587

To appear in: Received date: Revised date: Accepted date:

30-8-2015 28-10-2015 26-11-2015

Please cite this article as: Mendis, M., Leclerc, E., and Simsek, S.,Arabinoxylans, gut microbiota and immunity, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.11.068 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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Arabinoxylans, gut microbiota and immunity

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Mihiri Mendisa, Estelle Leclercb and Senay Simseka*

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Program, Fargo, ND,U.S.A.

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Professions, Fargo, ND, U.S.A.

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[email protected]

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North Dakota State University, Department of Plant Sciences, Cereal Science Graduate

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North Dakota State University, Department of Pharmaceutical Sciences, College of Health

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Corresponding author: Phone, 701-231-7737; Fax, 701-231-8474; E-mail,

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Arabinoxylan (AX) is a non-starch polysaccharide found in many cereal grains.

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The fermentation of AX by gut bacteria is influenced by its fine structure.

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The immunomodulatory properties of AX are also related to its fine structure.

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Abstract

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Arabinoxylan (AX) is a non-starch polysaccharide found in many cereal grains and is

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considered as a dietary fiber. Despite their general structure, there is structural heterogeneity

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among AX originating from different botanical sources. Furthermore, the extraction procedure

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and hydrolysis by xylolytic enzymes can further render differences to theses AX. The aim of this

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review was to address the effects of AX on the gut bacteria and their immunomodulatory

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properties. Given the complex structure of AX, we also aimed to discuss how the structural

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heterogeneity of AX affects its role in bacterial growth and immunomodulation. The existing

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literature indicates the role of fine structural details of AX on its potential as polysaccharides that

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can impact the gut associated microbial growth and immune system.

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Key words: Arabinoxylan, fine structure, immunomodulator, bacterial growth

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In the recent years there has been an increased interest in the area of dietary fibers in

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1. Introduction

food. This interest has been triggered by many health related properties associated with dietary

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fibers such as decreasing the risk for type 2 diabetes mellitus (T2D), cardiovascular disease, and

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colon cancer (Kaczmarczyk, Miller & Freund, 2012; Mudgil & Barak, 2013; Sorensen et al.,

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2014). An important dietary fiber is arabinoxylan (AX) which is the main non-starch

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polysaccharide in cereals (Stone, 2009). In most cereals the cell wall of endosperm cells and

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aleurone layer consists of about 60-70% arabinoxylans (Izydorczyk & Biliaderis, 1995). In wheat

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bran, arabinoxylan accounts for about 11-26% of wheat bran (Apprich et al., 2014). In this

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review we aimed to address the effects of AX on the gut bacteria and their immunomodulatory

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properties. Given the complex structure of AX, we also aim to discuss how the structural

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heterogeneity of AX affects its role in bacterial growth and immunomodulation.

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2. Arabinoxylan

In wheat grain, the major polymer of the cell wall is arabinoxylan (AX) (Saulnier, Sado,

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Branlard, Charmet & Guillon, 2007). Even though they occur as a minor constituent of the grain,

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due to the unique physico-chemical properties of AXs they play an important role in cereal food

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industry, including bread making (Courtin & Delcour, 2002), gluten-starch separation (Frederix,

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Van hoeymissen, Courtin & Delcour, 2004), refrigerated dough syruping (Courtin, Gys,

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Gebruers & Delcour, 2005; Simsek & Ohm, 2009) and in animal feeds (Bedford & Schulze,

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1998). However, despite their ability to exert physical changes to a system, they also possess the

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ability to exert biological changes to a system: AXs have been associated with beneficial health

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effect in patients with impaired glucose tolerance (Garcia et al., 2007). They observed that AX

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consumption improved postprandial metabolic responses in subjects with impaired glucose

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tolerance. AX extracted from wheat bran has been shown to have potent effects on innate and

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acquired immune response in mice (Cao et al., 2011). Many researches indicated the

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immunomodulatory properties of AX using a modified AX from rice bran (Gollapudi &

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Ghoneum, 2008; Pérez-Martínez et al., 2015; Zheng, Sugita, Hirai & Egashira, 2012).

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Although AX from different sources differ in their substitution along the xylan

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backbone, a general structure can be assigned for AX (Izydorczyk & Biliaderis, 1995). AX

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consists of a backbone of β-(1,4)-linked xylose residues, which are substituted with arabinose

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residues on the C(O)-2 and/or C(O)-3 position (Dornez, Gebruers, Delcour & Courtin, 2009).

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Phenolic acids such as ferulic acid, can be ester linked on the C(O)-5 position of arabinose.

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Under oxidative conditions, these ferulic acid residues undergo oxidative cross-linking forming

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inter/intra chain diferulic acid bridges (Geissman & Neukom, 1973). The structure of AX and

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enzymes involved in its degradation are shown in Figure 1 (adapted from Grootaert, Delcour,

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Courtin, Broekaert, Verstraete and Van de Wiele (2007).

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Two different classes of AX exist in wheat. The first class includes water-extractable

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AXs (WE-AXs), which account for about 25-30% of AXs in wheat flour and the second class

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consists of water-unextractable AXs (WU-AXs), which account for the remainder of AXs in

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wheat flour (Meuser & Suckow, 1986). WE-AXs are loosely bound to the cell wall surface

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(Mares & Stone, 1973). In contrast, WU-AXs are retained in the cell wall by covalent and non-

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covalent interactions with AXs and proteins, lignin and cellulose (Iiyama, Lam & Stone, 1994).

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AXs can be hydrolyzed by enzymatic and/or chemical means to produce AX hydrolyzates with 4 Page 4 of 27

varying length of the backbone (degree of polymerization, DP) and degree of substitution (DS).

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Thus, the structural details of an AX are dependent on its source, method of extraction, enzymes

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used for its hydrolysis, etc.

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2.1.

Endo-β-(1,4)-D-xylanases (xylanases)

Xylanases are the major enzymes involved in AX degradation. They cleave AXs by

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internally hydrolyzing the 1,4- β-D-xylosidic linkage between xylose residues in the xylan

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backbone in a random manner (Collins, Gerday & Feller, 2005; Dornez, Gebruers, Delcour &

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Courtin, 2009). Over 290 xylanases have been identified (Fierens, 2007) and have been grouped

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into six different glycoside hydrolase (GH) families (5, 7, 8, 10, 11 and 43) (Collins, Gerday &

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Feller, 2005; Dornez, Gebruers, Delcour & Courtin, 2009). The degradation pattern of each of

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these enzymes can be different, giving rise to different enzymatic products. For example, most of

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the glycoside hydrolases that are classified in the GH 10 family are endo-β-1,4-xylanases which

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degrade AX with high degree of substitution (DS) into smaller fragments (Pollet, Delcour &

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Courtin, 2010). GH 11 xylanases preferably cleave unsubstituted regions of the backbone and

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require three unsubstituted consecutive xylose residues for hydrolysis. Hence, GH 11 xylanases

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have a low activity on heteroxylans with a high degree of substitution (Pollet, Delcour &

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Courtin, 2010).

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2.2.

Arabinofuranosidases

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α-L-arabinofuranosidase (α-L-arabinofuranosidase arabinofuranohydrolase, EC 3.2.1.55,

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arabinofuranosidase) is an exo-enzymes that hydrolyze terminal nonreducing α-arabinofuranoses

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from arabinoxylans (Saha, 2000). Arabinofuranosidases have been classified into seven

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glycoside hydrolase families (GH 2, 3, 10, 43, 51, 54 and 62) (Lombard, Golaconda, Drula,

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Coutinho & Henrissat, 2014). Arabinofuranosidase from different families can have different

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substrate specificities. For example, arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from

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Bifidobacterium adolescentis (GH43) releases only C3-linked arabinose residues from double-

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substituted xylose residues (Van den Broek, Lloyd, Beldman, Verdoes, McCleary & Voragen,

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2005) while α-L-arabinofuranosidases from Clostridium thermocellum (GH51) catalyze the

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hydrolysis of α-1,3 arabinosyl substitutions of AX (Taylor, Smith, Turkenburg, D'souza, Gilbert

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& Davies, 2006).

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The structure of native AX itself is complex. Yet the degradation of this complex

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structure by a repertoire of xylanase and arabinofuranosidases with different substrate

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specificities, gives rise to an even complex cocktail of products. This is one of the reasons that

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make the identification of structure-biological response relationships difficult for AX. AXs are

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complex heteroxylans with different DP, DS and type of substitution molecule (Joseleau, Cartier,

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Chambat, Faik & Ruel, 1992; Pollet, Delcour & Courtin, 2010). The hydrolysis products of AX

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degradation (AX hydrolyzates) depend on the substrate specificity of the hydrolysis enzyme,

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giving rise to AX hydrolyzates with varying DP and DS. Thus, the intestinal microflora and the

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epithelial cells are exposed to an array of AX hydrolyzates with different fine structural details.

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Given the diversity of the native AX and the AX hydrolyzates, it is important to elucidate if there

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is a relationship between these structural details and their biological implications.

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The intestine is an important organ that consists of a huge surface area and permits vital

3. Human Gut Microbiota and AX

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interactions with micro-organisms living within the intestine, referred to as gut microbiota (Cani,

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Everard & Duparc, 2013; Walter & Ley, 2011). The gut microbiota exerts significant impacts on

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the host physiology, such as the control of energy homeostasis, the immune system, digestion 6 Page 6 of 27

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and vitamin synthesis (Cani, Everard & Duparc, 2013) and inhibition of pathogen colonization

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(Wardwell, Huttenhower & Garrett, 2011).

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The human diet is rich in plant and animal derived glycans such as AX. A large array of these glycans is resistant to digestion by human enzymes and relies on microbial enzymes for

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their digestion. The fermentation of these glycans by microbes yield energy for the microbial

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growth, and the end products such as short chain fatty acids (SCFA), mainly acetate, propionate

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and butyrate have profound effects on the health of the host (Tremaroli & Backhed, 2012).

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Butyrate acts mainly as the energy substrate for the colonic epithelium because it is the preferred

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energy source of colonocytes (Hamer, Jonkers, Venema, Vanhoutvin, Troost & Brummer, 2008;

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Koropatkin, Cameron & Martens, 2012). Acetate and propionate are absorbed into the blood

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stream and travel to the liver where they get incorporated into lipid and glucose metabolism,

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respectively (Rombeau & Kripke, 1990). Thus, acetate and propionate act as energy source to

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peripheral tissue cells. In addition to that, SCFA have an important effect on the host immune

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system as well. Low levels of butyrate modify the cytokine production profile of TH cells (Kau,

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Ahern, Griffin, Goodman & Gordon, 2011), promote intestinal epithelial barrier integrity and

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have also been associated with colonic tumor suppression (Hamer, Jonkers, Venema,

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Vanhoutvin, Troost & Brummer, 2008). In addition to being absorbed by the host, acetate is

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linked to maintaining the intestinal barrier function(Kau, Ahern, Griffin, Goodman & Gordon,

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2011), and preventing colonization of some enteric pathogens (Fukuda et al., 2011).

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The functional association among the intestinal microbiota, intestinal epithelial cells and

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the host immune system helps maintain the balance between tolerance and immunity to

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pathogenic or nonpathogenic microbes, or food ingredient. The fermentation of

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arabinoxylanoligosaccharides (AXOS) in vitro resulted in predominant production of acetate 7 Page 7 of 27

followed by propionate and butyrate (Snelders et al., 2014). The fermentability of AX by the

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intestinal microbiota is affected by the structural features of these AX. AX from different

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botanical sources has been shown to be fermented differently by the gut bacteria (Rose, Patterson

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& Hamaker, 2010). Among the maize, rice and wheat bran alkaline soluble fractions (principally

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AX), maize AX resulted in the highest SCFA production compared to fractions from rice and

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wheat in an in vitro fermentation system utilizing human fecal microbial cultures (Rose,

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Patterson & Hamaker, 2010). The fine structural differences among these three AX were related

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to the differences in fermentation indicating the importance of structure on the fermentability.

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They indicated that maize and rice bran AX were fermented by a debranching mechanism by the

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bacteria while wheat bran AX was degraded by a two phase mechanism; initially degrading the

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unsubstituted regions followed by the fermentation of highly branched regions (Rose, Patterson

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& Hamaker, 2010). Rice AX was shown to be more highly branched compared to maize AX,

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thus, the bacteria were required to remove the arabinose from the AX before being able to utilize

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xylose in the case of rice AX. The maize AX was comparatively less branched and the relative

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ease of reaching xylose could have resulted in the increased SCFA production in maize AX. In a

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similar experiment using in vitro human fecal batch experiments, it was shown that there was no

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relationship of molecular mass, arabinose:xylose ratio, or degree of substitution to fermentation

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rate patterns (Rumpagaporn, Reuhs, Kaur, Patterson, Keshavarzian & Hamaker, 2015).

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However, fast fermenting rice and sorghum AX was shown to have simple branched structure

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compared to what and maize AX (Rumpagaporn, Reuhs, Kaur, Patterson, Keshavarzian &

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Hamaker, 2015). Furthermore, ferulic acid substitution on the AX oligosaccharides was shown to

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hinder fermentation (Snelders et al., 2014). Upon in vitro fermentation of AXOS, AXOS that

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were highly feruloylated were shown to be less fermented compared to less feruloylated AXOS

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(Snelders et al., 2014). Thus, it is evident that the fine structural details of AX play an important

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role in governing its fermentation.

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AXs are one of the most abundant polysaccharide in cell walls of cereals and cereals being a large proportion of the human diet, large quantities of AXs are introduced to the gastro

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intestinal tract (GIT). Healthy adult gut microbiota is composed primarily of members of two

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bacterial phyla, the Bacteroidetes and Firmicutes (McNulty et al., 2013). However, the most

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expanded glycolytic gene collection that target xylan degradation is possessed by genus

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Bacteroides (Zhang et al., 2014). The members of the genus Bacteroides are adept at utilizing

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plant and host derived polysaccharides (Koropatkin, Cameron & Martens, 2012). These

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Bacteroides are rich in genes involved in the acquisition and metabolism of various glycosides

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including glycoside hydrolases and polysaccharide lyases. These enzymes are organized into

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polysaccharide utilization loci (PULs) that are distributed throughout the genome(Koropatkin,

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Cameron & Martens, 2012). In the phylum Bacteroidetes, the metabolism of starch from the

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external environment is achieved via the starch utilization system (Sus) (Koropatkin, Cameron &

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Martens, 2012). Similar systems targeting the degradation of non-starch glycans have also been

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described in the Bacteroidetes phylum and have been therefore named Sus-like systems

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(Koropatkin, Cameron & Martens, 2012). The molecular mechanisms used to utilize xylan have

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been suggested to be analogous to the Sus-like paradigm (Dodd, Mackie & Cann, 2011; Martens,

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Koropatkin, Smith & Gordon, 2009). For example, a gene cluster induced in the presence of

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wheat xylan was recently identified in Bacteroidetes and termed the xylan utilization system

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(Xus) (Dodd, Mackie & Cann, 2011). The Xus consists of a set of polysaccharide binding

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proteins, glycolytic enzymes that hydrolyze large polysaccharides into smaller oligosaccharides

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and TonB-dependent transporters that transport these oligosaccharides into the periplasm (Zhang

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et al., 2014). These oligosaccharides are then converted to smaller monosaccharides by an array

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of glycolytic enzymes before being transported to the cytosol (Martens, Koropatkin, Smith &

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Gordon, 2009). The biochemical pathway for xylose metabolism is the pentose phosphate

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pathway (PPP)(Jeffries, 2006). There are two main routes of how xylan enters the PPP; the

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xylose isomerase pathway, which is common among bacteria, and the redox pathway, which is

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common among eukaryotes. In the bacterial pathway, xylose is converted to xylulose by the

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action of xylose isomerases and then phosphorylated to xylulose-5-phosphate by xylulokinases

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before entering the PPP (Dodd, Mackie & Cann, 2011; Jeffries, 2006).

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Apart from Bacteroidetes many other bacterial phyla also possess the enzymes capable of degrading AX (Grootaert, Delcour, Courtin, Broekaert, Verstraete & Van de Wiele, 2007). The

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presence of arabinoxylan degrading enzymes in these bacterial species indicates the association

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between AX and survival of these bacterial species in the gut. The ability of arabinoxylan

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oligosaccharides to promote the growth of some lactobacilli has been reported (Kontula, Suihko,

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Suortti, Tenkanen, Mattila-Sandholm & von Wright, 2000). Using mixed culture fermentation

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systems Vardakou, Palop, Christakopoulos, Faulds, Gasson and Narbad (2008) evaluated the

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effect of AX derived from wheat on the modulation of gut bacterial composition. They observed

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an increase in the prebiotic index with AX treated with xylanase compared to AX that did not

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receive xylanase treatment indicating the effect of DP on the fermentability of the specific AX.

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Thus, the possibility of using arabinoxylan as a substrate in a symbiotic product containing

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probiotic bacteria has been suggested (Vardakou, Palop, Christakopoulos, Faulds, Gasson &

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Narbad, 2008).

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Xylooligosaccharides have been shown to increase the total cecal weight and bifidobacteria population in rats (Hsu, Liao, Chung, Hsieh & Chan, 2004). These 10 Page 10 of 27

xylooligosaccharides performed better than the commonly used fructooligosaccharides with

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respect to their beneficial health effects (Hsu, Liao, Chung, Hsieh & Chan, 2004). Research

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using fish (juvenile Siberian sturgeon (Acipenser baerii)) fed with diet containing 2% AXOS

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showed that AXOS mainly stimulated the growth of lactic acid bacteria and Clostridium sp.

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(Geraylou et al., 2012). More so, AXOS with higher DP (AXOS-32-0.30; (average DP 32;

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average DS 0.30) were more effective in stimulating the growth of these bacteria compared to

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AXOS with lower DP (AXOS-3-0.25) (Geraylou et al., 2012). They observed that the AXOS

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improved the health of the fish through prebiotic action and that the AXOS with higher DP had

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better health promoting effects compared to the AXOS with lower DP. Jaskari, Kontula,

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Siitonen, Jousimies-Somer, Mattila-Sandholm and Poutanen (1998) observed that

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Bifidobacterium sp. favored xylooligomers to hexose sugars as substrate. They also speculated

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that the content of arabinose substitution along the xylooligosaccharide backbone might

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determine the selectivity for these oligosaccharides by Bifidobacterium and Bacteroides sp. with

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substrates with high arabinose substitution being favored by Bifidobacterium compared to

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Bacteroides sp. However, overall better fermentability was observed for AX with lower

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arabinose substitution compared to AX with higher arabinose substitution (Amrein, Gränicher,

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Arrigoni & Amadò, 2003).

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Neyrinck et al. (2011) observed that AX supplementation was effective in restoring the

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microbial shifts induced by high fat diets in mice. Furthermore, AX caused a specific increase in

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Bifidobacteria, specifically Bifidobacterium animalis ssp lactis indicating the prebiotic potential

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of AX. The effect of long chain AX on short chain fatty acid production and Bifidobacteria

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composition has been investigated using in vitro gut models (Van den Abbeele, Venema, Van de

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Wiele, Verstraete & Possemiers, 2013). They detected that long chain AX increased the 11 Page 11 of 27

Bifidobacterium longum population and propionate levels. Spreading the availability of

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fermentable substrates to the distal colon is an important aspect to counteract the toxic

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environment in the distal colon. Compared to ‘gold-standard’ prebiotics such as inulin which is

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mainly fermented in the ascending colon, long chain AX can extend to consecutive regions of the

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colon to get fermented (Van den Abbeele, Venema, Van de Wiele, Verstraete & Possemiers,

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2013). Thus, long chain AX need to be further evaluated as a prebiotic that can extend to the

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distal colon regions.

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These data indicate the importance of paying close attention to the fine structural

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differences of AX rather than considering only the general structure of AX when evaluating AX

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for its biological properties. The specific relationship between the fine structural details of AX

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and gut microbial composition is an area that needs to be explored further. Such insight might

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shed light on the development of dietary fibers with specific structural details with the aim of

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altering the specific gut microbial population to achieve desired outcomes.

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4. Immunological Effects of AX

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4.1.

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The cells of the innate immune system are first line of defense against microbes and

Recognition of dietary constituents by immune system

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infectious agents that are encountered by the body (Delcenserie, Martel, Lamoureux, Amiot,

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Boutin & Roy, 2008). The components of the innate immune system consist of epithelial

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barriers, phagocytes (neutrophils, monocytes and macrophages), dendritic cells, natural killer

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(NK) cells, complement proteins, cytokines and other types of proteins.

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The cells of the innate immune system consist of receptors that recognize structures that are non-self and that are not present in the host cells (Abbas & Lichtman, 2011). Specific 12 Page 12 of 27

molecules present at the surface of microbes are targeted by the innate immune system and are

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termed microbe-associated molecular patterns (MAMPs) or pathogen associated molecular

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patterns (PAMPs). The receptors of the innate immune system that recognize these foreign

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components are called pattern recognition receptors and include receptors of the toll like receptor

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(TLR) family and mannose receptors. TLRs are involved in the recognition of many bacterial

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lipopolysaccharides.

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Recognition of PAMPs by receptors of the innate immune system results in a

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cascade of cellular events that bring about effector functions to eliminate the threat. For instance,

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activation of TLRs by PAMPs involves cellular processes such as recruitment and activation of

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protein kinases which leads to the activation of transcription factors such as nuclear factor кB

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(NF-кB) and interferon response factor-3 (IRF-3), which direct the gene transcription of

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inflammatory cytokines (e.g. interleukin (IL)-1, IL-8), enzymes and proteins involved in

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eliminating the microbes. However, the function of the innate immune system is not restricted to

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the initial defense against infection. The innate immune system also plays an important role in

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instructing the adaptive immunity to activate its response. Although the exact receptors for AX

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have not yet been identified it is speculated that TLR4 might be a possible candidate (Zhang, Li,

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Smith & Musa, 2014). The structural resemblance of rice bran and corn husk derived AX to the

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bacterial lipopolysaccharide in terms of structure and molecular weight supports this speculation

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(Ogawa, Takeuchi & Nakamura, 2005; Otterlei, Sundan, Skjak-Braek, Ryan, Smidsrod &

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Espevik, 1993; Zhang, Li, Smith & Musa, 2015). Moreover, TLR2 and TLR6 could also be

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involved in recognition of AX by cells (Volman, Ramakers & Plat, 2008).

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The intestine is exposed to wide range of dietary fibers including AX that might be present in the diet. The immune response of the intestinal mucosa is mediated by specialized 13 Page 13 of 27

features associated with the intestinal mucosa. It consists of Peyer’s patches, isolated lymphoid

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follicles (ILFs) and mesenteric lymph nodes (MLNs) (Maynard, Elson, Hatton & Weaver, 2012)

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as indicated in Figure 2(adapted from Aimutis (2011)). Peyer’s patches are lymphoid follicle

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aggregates which contain T and B lymphocytes, plasma cells, macrophages and dendritic cells

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(Langkamp-Henken, Glezer & Kudsk, 1992). M-cells (microfold cells) are specialized epithelial

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cells that overlie the Peyer's patches (Kagnoff, 1993; Schley & Field, 2002). M-cells are

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involved in endocytosis and transport of antigens from the intestinal lumen to the Peyer’s patches

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where they are presented to T- and B-lymphoctes (Kagnoff, 1993; Langkamp-Henken, Glezer &

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Kudsk, 1992). Activation of B-lymphocytes following antigen presentation leads to the

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production of antibodies. Also, the activated immune cells exit Peyer’s patches and enter the

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systemic circulation. Thus, the Peyer’s patches act as the main sampling site for ingested

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antigens (Schley & Field, 2002). T-and B-lymphocytes, plasma cells, mast cells and

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macrophages are distributed throughout the lamina propria (Langkamp-Henken, Glezer &

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Kudsk, 1992). Interepithelial lymphocytes are spread on the interstitial spaces of the epithelial

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cells and are in direct contact with intestinal antigens suggesting that they might be the first cells

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of the immune system that interact with and respond to ingested antigens (Schley & Field, 2002).

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MLNs comprise of the immune cells entering and leaving the gut (Schley & Field, 2002). ILFs

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are similar to Peyer’s patches but they mainly consist of B-cells, dendritic cells and the overlying

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epithelium comprising of M cells. (Lamichhane, Azegami & Kiyono, 2014; Tsuji et al., 2008).

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They are proposed to be involved in the production of T cell independent IgA production (Tsuji

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et al., 2008).

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4.2.

AXs as immunomodulators

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A possible role of dietary polysaccharides, apart from being fermented by intestinal microbiota, is associated with its immunological effects. Evidence suggests that addition of fiber

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to the diet alters the structure and function of the gut, and modifies the gut-derived hormones and

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production of cytokines (Mikkelsen, Jespersen, Mehlsen, Engelsen & Frokiaer, 2014; Schley &

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Field, 2002). Most of the research on this area has been performed with beta-glucans while other

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cereal polysaccharides such as arabinoxylans are starting to gain interest as possible

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immunomodulators. Immunomodulators or biologic response modifiers are compounds that

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interact with the host immune system and bring about upregulation or downregulation of specific

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immune responses (Tzianabos, 2000). Experimental work performed in vitro with monoculture

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cell lines have suggested that polysaccharides are capable of directly stimulating the intestinal

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epithelial cells and monocytes, thereby bringing about immunological outcomes (Chan, Chan &

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Sze, 2009; Mikkelsen, Jespersen, Mehlsen, Engelsen & Frokiaer, 2014; Rieder & Samuelsen,

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2012; Volman, Ramakers & Plat, 2008; Xu, Xu, Ma, Tang & Zhang, 2013). If AX behaves

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similarly to dietary fibers such as beta-glucan, they could also exert similar immunological

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outcomes upon interaction with intestinal epithelial cells.

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Inflammation is an adaptive response that is triggered by noxious stimuli and conditions,

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such as infection and tissue injury (Medzhitov, 2008). Under healthy physiological conditions

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inflammation is a defense response that serves a protective function in the body. Although the

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exact mechanism of how the dietary fibers affect the immune response and inflammation is not

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well understood, several mechanisms have been studied (Schley & Field, 2002). Depending on

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their structure, dietary fibers can be taken up by M-cells in the Peyer’s patches and be

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transported to underlying immune cells and other cells which results in local cytokine production

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(Volman, Ramakers & Plat, 2008). This in turn will influence T-cells, B-cells, antigen presenting 15 Page 15 of 27

cells and other immune cells. Fibers may also be taken up by intestinal macrophages and

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dendritic cells and transported to lymph nodes, spleen and bone marrow. Moreover, direct

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interaction of fibers with colonic epithelial cells or leukocytes may induce changes in immune

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reactions related to inflammation and development of cancer (Samuelsen, Rieder, Grimmer,

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Michaelsen & Knutsen, 2011; Volman, Ramakers & Plat, 2008).

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Animal studies have clearly demonstrated that dietary fiber type and content can modulate measures of immune function. Partially hydrolyzed water-soluble corn husk

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arabinoxylan (average molecular weight of about 53 kDa) is shown to have immunological

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outcomes after oral administration in mice (Ogawa, Takeuchi & Nakamura, 2005). This anti-

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inflammatory action of corn husk AX is thought to be through activation of an INF-ɣ dependent

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T helper 1-like immune response (Ogawa, Takeuchi & Nakamura, 2005)

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AXs extracted from wheat bran have been shown to have potent effects on innate and

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acquired immune response in mice (Cao et al., 2011). They observed that oral administration of

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wheat bran derived AX significantly inhibited the growth of transplantable tumors and promoted

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the NK cell and macrophage phagocytosis activity in S180 tumor-bearing mice (Cao et al.,

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2011). The study also indicated that AX increased the IL-2 in blood serum in a dose dependent

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manner (Cao et al., 2011). However, they also indicated that the AX did not inhibit tumor cells in

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vitro which suggest that the anti-tumor activity of AX was related to immunostimulatory

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properties. Thus, they suggested that AX extracted from wheat bran by alkaline or enzymatic

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procedures could be a good source of natural immunomodulation. AXs significantly increase the

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activation potential of T and B cells and enhance the humoral and cell-mediated immunity in

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tumor bearing mice. Akhtar et al. (2012) demonstrated that wheat bran derived AXs have the

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potential to stimulate antibody-mediated immune response in chickens. Thus, AXs have the

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16 Page 16 of 27

potential as immuno-enhancing and antioxidant additives in functional foods (Hromádková,

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Paulsen, Polovka, Košťálová & Ebringerová, 2013). MGN-3/Biobran, a rice bran AX that has

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been modified using mushroom derived enzymes (Ghoneum & Abedi, 2004), is one of the most

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studied AX in relation to their immunological properties (Badr El-Din, Noaman, Ghoneum &

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Ghoneum, 2007; Ghoneum & Abedi, 2004; Ghoneum & Gollapudi, 2003, 2006; Gollapudi &

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Ghoneum, 2008; Noaman, Badr El-Din, Bibars, Abou Mossallam & Ghoneum, 2008; Pérez-

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Martínez et al., 2015). The effects of MGN-3 on macrophage function was examined in vitro

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using 3 macrophage cell lines; human macrophage cell line U937, murine macrophage cell line

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RAW264.7, and murine peritoneal macrophages (P-M phi) (Ghoneum & Matsuura, 2004). The

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treatment of macrophages with MGN-3 increased the attachment and phagocytosis of yeast by

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macrophages in a dose dependent manner. Enhancement in the spreading ability of macrophages

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and increased nitric oxide production was also observed upon treatment with MGN-3(Ghoneum

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& Matsuura, 2004). MGN-3 also significantly increased the production of TNF-alpha; and IL-6

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in a dose dependent manner (1, 10,100 µg/ml) in macrophages. They concluded that MGN-3 is a

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potent inducer of phagocytic function by macrophage and suggested a role for MGN-3 as an

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agent that could be employed in the fight against microbial infection (Ghoneum & Matsuura,

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2004). A study carried out to evaluate the effect of MGN-3 in enhancing the natural killer (NK)

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cell activity in aged C57BL/6 and C3H mice upon intraperitoneal injection of MGN-3 (10 mg

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kg−1 per day) resulted in a drastic increase in the peritoneal NK activity in mice(Ghoneum &

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Abedi, 2004). It was suggested that MGN-3 enhances murine NK activity of aged mice and may

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be useful for enhancing NK function in aged humans (Ghoneum & Abedi, 2004). These

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researches reinforce the credibility of AXs as immunomodulators. However, considering the

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structural complexity of AX molecule, the contribution from the fine structural variations in AX

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to these immunomodulatory properties cannot be ignored. The immunological properties of the polysaccharides depend on its fine structure.

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(Adams et al., 2008; Mikkelsen, Jespersen, Mehlsen, Engelsen & Frokiaer, 2014). Depending on

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the extraction method, AX can exhibit structural differences. The effect of extraction method on

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the immunological outcomes of AX has been evaluated (Zhou, Liu, Guo, Wang, Peng & Cao,

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2010). They obtained alkaline extracted AX (AXA) and enzyme-extracted AX (AXE) from wheat

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bran which were shown to have different structural compositions. AXA was highly substituted

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with arabinose with an arabinose to xylose ratio of 0.83 compared to AXE which was less

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substituted (arabinose to xylose of 0.56). Also, the weight-average molecular weight of AXA

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(3.517 × 105 Da) was about 10 times that of AXE (3.252 × 104 Da). Despite their structural

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differences both AXA and AXE had potent stimulating effects on innate and acquired immune

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responses on oral administration in female BALB/c mice while AXE stimulated macrophage

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phagocytosis and delayed hypersensitivity reaction than did AXA (Zhou, Liu, Guo, Wang, Peng

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& Cao, 2010). However, interperitonial adiministration nor in vitro treatemnt with AXA or AXE

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confered significant immunostimulating effects (Zhou, Liu, Guo, Wang, Peng & Cao, 2010). The

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inability of AX to exert immunomodulatory effects in vitro as evident in studies by Cao et al.

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(2011) and Zhou, Liu, Guo, Wang, Peng and Cao (2010) indicate that the immunomodulatory

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properties of AX might be related to their in vivo metabolisum (Cao et al., 2011; Zhou, Liu, Guo,

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Wang, Peng & Cao, 2010).

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When reviewing the immunomodulatory properties of AX, the botanical source of AX

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also need to be considered. Immunostimulating activity of wheat bran derived AX was found to

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be higher than that for AX derived from corn husk or rice bran (Monobe, Maeda-Yamamoto, 18 Page 18 of 27

Matsuoka, Kaneko & Hiramoto, 2008). Oral administration of corn husk AX has been shown to

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significantly increase the interleukin (IL)-2 and interferon (IFN)-γ production with a slight

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increase in IL-4 in mitogen-induced proliferation of spleen cells in healthy mice (Ogawa,

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Takeuchi & Nakamura, 2005). The ability of corn husk AX (average molecular weight of about

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53 kDa) to elevate the immunopotentiating activity without causing overexpression of

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immunological reactions has also been suggested (Ogawa, Takeuchi & Nakamura, 2005). The

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effect of modified rice bran AX on the immune system was investigated by Ghoneum and

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Matsuura (2004) using three different macrophage cell lines in vitro (human macrophage cell

400

line U937, murine macrophage cell line RAW264.7, and murine peritoneal macrophages (P-M

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phi). They found that AX significantly induced production of TNF-alpha and IL-6 in a dose

402

dependent manner and also increased nitric oxide production significantly. This indicate the

403

potential of AX to induce phagocytic function in macrophages and thus, its ability to help fight

404

against microbial infection (Ghoneum & Matsuura, 2004).

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The immunological properties of AX also depend on molecular weight, chemical

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composition and substituted degree or branch of arabinose (Zhou, Liu, Guo, Wang, Peng & Cao,

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2010). Thus, the structure driven immunological properties need to be further evaluated.

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5. Conclusions

Depending on its botanical source, extraction method and chemical/enzymatic treatments,

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AX can have different structural complexities to begin with. Many gut microbes have evolved to

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contain enzymes, receptors and transporters that achieve efficient degradation and utilization of

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these complex AX molecules. Due to their variation in DP, DS, and ferulic acid substitution AX

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hydrolyzates are considerably diverse and complex molecules compared to many other dietary

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fibers. Our review demonstrats that just as AX plays a role in altering the microbial composition 19 Page 19 of 27

in the gut and exerting immunological outcomes, the role its fine structural details play in

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governing these effects are not to be undermined. Due to the compelling evidence on the

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biological properties of AX, further research that explores these properties and safety of AX is

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encouraged. However, given the complexity of different AX molecules, exploring the biological

419

capabilities of AX with emphasis on its specific fine structure rather than considering it as a

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generalized AX structure, is highly beneficial in unwrapping the true potential of AX

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Rose, D. J., Patterson, J. A., & Hamaker, B. R. (2010). Structural differences among alkali-soluble arabinoxylans from maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum) brans influence human fecal fermentation profiles. Journal of Agricultural and Food Chemistry, 58(1), 493-499. Rumpagaporn, P., Reuhs, B. L., Kaur, A., Patterson, J. A., Keshavarzian, A., & Hamaker, B. R. (2015). Structural features of soluble cereal arabinoxylan fibers associated with a slow rate of in vitro fermentation by human fecal microbiota. Carbohydrate Polymers, 130, 191-197. Saha, B. C. (2000). Alpha-L-arabinofuranosidases: biochemistry, molecular biology and application in biotechnology. Biotechnol Adv, 18(5), 403-423. Samuelsen, A. B., Rieder, A., Grimmer, S., Michaelsen, T. E., & Knutsen, S. H. (2011). Immunomodulatory activity of dietary fiber: arabinoxylan and mixed-linked beta-glucan isolated from barley show modest activities in vitro. International journal of molecular sciences, 12(1), 570-587. Saulnier, L., Sado, P. E., Branlard, G., Charmet, G., & Guillon, F. (2007). Wheat arabinoxylans: Exploiting variation in amount and composition to develop enhanced varieties. Journal of Cereal Science, 46(3), 261-281. Schley, P. D., & Field, C. J. (2002). The immune-enhancing effects of dietary fibres and prebiotics. British Journal of Nutrition, 87(S2), S221-S230. Simsek, S., & Ohm, J. B. (2009). Structural changes of arabinoxylans in refrigerated dough. Carbohydrate Polymers, 77(1), 87-94. Snelders, J., Olaerts, H., Dornez, E., Van de Wiele, T., Aura, A.-M., Vanhaecke, L., Delcour, J. A., & Courtin, C. M. (2014). Structural features and feruloylation modulate the fermentability and evolution of antioxidant properties of arabinoxylanoligosaccharides during in vitro fermentation by human gut derived microbiota. Journal of Functional Foods, 10, 1-12. Sorensen, M. D., Hsi, R. S., Chi, T., Shara, N., Wactawski-Wende, J., Kahn, A. J., Wang, H., Hou, L., & Stoller, M. L. (2014). Dietary Intake of Fiber, Fruit and Vegetables Decreases the Risk of Incident Kidney Stones in Women: A Women’s Health Initiative Report. The Journal of Urology, 192(6), 1694-1699. Stone, B. M., M. K. . (2009). Carbohydrates. In K. K. P. R. Shewry (Ed.). Wheat Chemistry and Technology (pp. 299-362). Minnesota: AACC International. Taylor, E., Smith, N., Turkenburg, J., D'souza, S., Gilbert, H., & Davies, G. (2006). Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum. Biochem.J, 395, 31-37. Tremaroli, V., & Backhed, F. (2012). Functional interactions between the gut microbiota and host metabolism. Nature, 489(7415), 242-249. Tsuji, M., Suzuki, K., Kitamura, H., Maruya, M., Kinoshita, K., Ivanov, I. I., Itoh, K., Littman, D. R., & Fagarasan, S. (2008). Requirement for Lymphoid Tissue-Inducer Cells in Isolated Follicle Formation and T Cell-Independent Immunoglobulin A Generation in the Gut. Immunity, 29(2), 261-271. Tzianabos, A. O. (2000). Polysaccharide immunomodulators as therapeutic agents: structural aspects and biologic function. Clin Microbiol Rev, 13(4), 523-533. Van den Abbeele, P., Venema, K., Van de Wiele, T., Verstraete, W., & Possemiers, S. (2013). Different human gut models reveal the distinct fermentation patterns of Arabinoxylan versus inulin. J Agric Food Chem, 61(41), 9819-9827. Van den Broek, L., Lloyd, R., Beldman, G., Verdoes, J., McCleary, B., & Voragen, A. (2005). Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083. Applied Microbiology and Biotechnology, 67(5), 641-647. Vardakou, M., Palop, C. N., Christakopoulos, P., Faulds, C. B., Gasson, M. A., & Narbad, A. (2008). Evaluation of the prebiotic properties of wheat arabinoxylan fractions and induction of

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hydrolase activity in gut microflora. International Journal of Food Microbiology, 123(1–2), 166170. Volman, J. J., Ramakers, J. D., & Plat, J. (2008). Dietary modulation of immune function by beta-glucans. Physiology & behavior, 94(2), 276-284. Walter, J., & Ley, R. (2011). The Human Gut Microbiome: Ecology and Recent Evolutionary Changes. Annual Review of Microbiology, 65(1), 411-429. Wardwell, L. H., Huttenhower, C., & Garrett, W. S. (2011). Current concepts of the intestinal microbiota and the pathogenesis of infection. Current infectious disease reports, 13(1), 28-34. Xu, X., Xu, P., Ma, C., Tang, J., & Zhang, X. (2013). Gut microbiota, host health, and polysaccharides. Biotechnology Advances, 31(2), 318-337. Zhang, M., Chekan, J. R., Dodd, D., Hong, P.-Y., Radlinski, L., Revindran, V., Nair, S. K., Mackie, R. I., & Cann, I. (2014). Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes. Proceedings of the National Academy of Sciences, 111(35), E3708-E3717. Zhang, S., Li, W., Smith, C. J., & Musa, H. (2014). Cereal-Derived Arabinoxylans as Biological Response Modifiers: Extraction, Molecular Features, and Immune-Stimulating Properties. Critical Reviews in Food Science and Nutrition, 55(8), 1035-1052. Zhang, S., Li, W., Smith, C. J., & Musa, H. (2015). Cereal-derived arabinoxylans as biological response modifiers: extraction, molecular features, and immune-stimulating properties. Crit Rev Food Sci Nutr, 55(8), 1035-1052. Zheng, S., Sugita, S., Hirai, S., & Egashira, Y. (2012). Protective effect of low molecular fraction of MGN-3, a modified arabinoxylan from rice bran, on acute liver injury by inhibition of NF-κB and JNK/MAPK expression. International Immunopharmacology, 14(4), 764-769. Zhou, S., Liu, X., Guo, Y., Wang, Q., Peng, D., & Cao, L. (2010). Comparison of the immunological activities of arabinoxylans from wheat bran with alkali and xylanase-aided extraction. Carbohydrate Polymers, 81(4), 784-789.

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Figure 1. Structure of arabinoxylan (AX) and arabinoxylan degrading enzymes (adapted from

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Grootaert, Delcour, Courtin, Broekaert, Verstraete & Van de Wiele (2007)). The backbone of

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AX is composed of β-(1,4)-linked xylose residues, which can be substituted with arabinose

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residues on the C(O)-2 and/or C(O)-3 position. Ferulic acid can be esterified on the C(O)-5

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position of arabinose. Endo-β-(1,4)-d-xylanases cleave the xylan backbone internally, β-d-

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xylosidases remove xylose monomers from the non-reducing end of xylo-oligosaccharides, α-l-

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arabinofuranosidases remove arabinose substituents from the xylan backbone, and ferulic acid

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esterases remove ferulic acid groups from arabinose substituents (adapted from Dornez,

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Gebruers, Delcour & Courtin (2009)).

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Figure 2. The gut associated lymphoid tissue (GALT) (adapted from Aimutis (2011)).Intestinal

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epithelial cells maintain a barrier between the contents of the intestinal lumen and the underline

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immune cells. APC: antigen presenting cells; M-cells: microfold cells; IgA: immunoglobulin A.

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