The functionality of prebiotics as immunostimulant: Evidences from trials on terrestrial and aquatic animals

Accepted Manuscript The functionality of prebiotics as immunostimulant: Evidences from trials on terrestrial and aquatic animals Asad Nawaz, Allah Bakhsh javaid, Sana Irshad, Seyed Hossein Hoseinifar, Hanguo Xiong PII:

S1050-4648(18)30121-9

DOI:

10.1016/j.fsi.2018.03.004

Reference:

YFSIM 5159

To appear in:

Fish and Shellfish Immunology

Received Date: 13 December 2017 Revised Date:

21 February 2018

Accepted Date: 2 March 2018

Please cite this article as: Nawaz A, Bakhsh javaid A, Irshad S, Hoseinifar SH, Xiong H, The functionality of prebiotics as immunostimulant: Evidences from trials on terrestrial and aquatic animals, Fish and Shellfish Immunology (2018), doi: 10.1016/j.fsi.2018.03.004. 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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Fig. 1. Graphical Abstract

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The functionality of prebiotics as immunostimulant: evidences from trials on

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terrestrial and aquatic animals Authors: Asad Nawaz1, Allah Bakhsh javaid1, Sana Irshad2, Seyed Hossein Hoseinifar3*,

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Hanguo Xiong1**

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College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

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School of Environmental studies, China University of Geosciences, Wuhan 430070, China

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Department of Fisheries, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan,

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Iran

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*co-corresponding author: Seyed Hossein Hoseinifar

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**corresponding author: Hanguo Xiong [email protected]

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Abstract: The gut immune system is, the main option for maintaining host’s health, affected by numerous

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factors comprising dietary constituents and commensal bacteria. These dietary components that

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affect the intestinal immunity and considered as an alternative of antibiotics are called

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immunosaccharides. Fructooligosaccharide (FOS), Galactooligosaccharide (GOS), inulin, dietary

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carbohydrates, and xylooligosaccharide (XOS) are among the most studied prebiotics in human

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as well as in aquaculture. Although prebiotics and probiotics have revealed potential as treatment

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for numerous illnesses in both human and fish, a comprehensive understanding of the molecular

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mechanism behind direct and indirect effect on the intestinal immune response will help more

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and perhaps extra effective therapy intended for ailments. This review covers the most newly

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deep-rooted scientific outcomes about the direct and indirect mechanism through which these

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dietetic strategies can affect intestinal immunity of terrestrial and aquatic animals. Prebiotics

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exert an influence on gut immune system via the increase in lysozyme and phagocytic activity,

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macrophage activation and stimulation of monocyte-derived dendritic cells. Furthermore, these

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functional molecules also enhance epithelial barrier function, beneficial gut microbial

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population, and production of intermediate metabolites for example short chain fatty acids

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(SCFAs) that assist in balancing the immune system. Moreover, emphasis will be sited on the

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relationship among food/feed, the microbiota, and the gut immune system. In conclusion, further

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studies are nonetheless essential to confirm the direct effect of prebiotics on immune response.

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Key words: Prebiotics, Probiotics, Immunity, SCFA, Mechanism of action,

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Abbreviations: Gastro intestinal track (GIT), gut-associated lymphoid tissue (GALT), pattern

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recognition receptor (PRR), Toll-like receptors (TLR), Fructose oligosaccharides (FOS),

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galactose oligosaccharides (GOS)

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1. Introduction

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Prebiotics are functional foods that have multiple beneficial effects on the host [1]. The

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concept of prebiotics as a functional food and its benefit to vertebrate gut microbiota was first

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defined as “a non-digestible food ingredient that beneficially affects the host by selectively

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stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and

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thus improves host health”[2]. The current definition of prebiotics has been suggested by [3],

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“Prebiotics are food constituents that well thought-out to be non-digestible selectively fermented,

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confers benefits of growth and/or activity of beneficial microbes present in gastrointestinal tract

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and improve the health of host”. To be considered as prebiotics, these must be: (i) resistant to

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stomach acidity, mammalian enzymes and gastrointestinal assimilation (ii) fermentable by

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beneficial intestinal microbes (iii) capable to encourage the escalation and/or action of special

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microbes linked with health and welfare of the host. In this regard, the most recognized

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prebiotics are Fructose oligosaccharides (FOS), Galactose oligosaccharides (GOS), inulin,

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polydextrose, xylose oligosaccharides (XOS), some dietary fibers, and mannan-oligosaccharide

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[1]. As food or feed ingredients, prebiotics confer many health benefits on human and fish

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including immune stimulation, intestinal mucosal morphology [4], promoting epithelial barrier,

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and phagocytosis [5-7]. The most important valuable effects of prebiotics are obtained through

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their impact on the gastrointestinal tract and immune system of fish and human [1, 8].

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The immune system is a first weapon against infections (entry of microbes) and toxins

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(produced in the body or uptake through the food, feed, water or environment), and its function

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is highly dependent on provision of adequate nutrients. In the GIT trillion of microbes compete

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for nutrition and attachment sites which results in strong competition between beneficial and

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pathogenic microbes [9]. Beneficial microbes are able to produce some antimicrobial agents for

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example bacteriocins and short chain fatty acids (SCFA) that can promote the immune response

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[10, 11]. Hydrolysis/fermentation of prebiotics by microbes result in different metabolites that

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give extra energy to the cell [12]. Gut microbes are highly dependent on food ingested by the

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host. Food/feed also plays a vital role in balancing beneficial and pathogenic microbes due to

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their digestive (polysaccharides) nature [13]. Microbes differ with the age group, sex, health

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status, genetic factor, environmental conditions and even season to season. It is well known that

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from early age or first day of feeding numbers of microbes are less than the elderly ages or

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adulthood. It is because of alteration of diet, body size, environmental exposure and revelation of

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disease with the passage of time [13]. Provision of prebiotics alters the immune response and

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reduces risk of disease in a new born baby [14]. Recommendations of prebiotics in early life

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stage, during pregnancy and even after birth can be considered to improve the composition of gut

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microbes as preventive measure against the infection and modulation of immune system [15, 16].

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Advanced studies on gut microbes and their broad diversity have helped us to learn the

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mechanism; how microbes digest food and their metabolites influence the host immune

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mechanism [17].

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The gastrointestinal immune system has the inimitable capacity to differentiate between

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beneficial and potentially dangerous material. It can promote protective response against harmful

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microbes and toxins while accepting food antigens and commensal microbes. Direct influence of

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prebiotics on immune system is through activation of cellular receptors [18, 19]. The prebiotics

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that directly influence the immune system are further classified as immunosaccharides or

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immunostimulants [19]. The gut epithelial cell release some antimicrobial protein that acts as

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natural antibiotics belong to the proteins families that directly kills the harmful microbes by the

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action on the cell wall [20, 21]. Prebiotics stimulate these epithelial cells to produce a high

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amount of antimicrobial compound [22]. This helps the host to bear a significant number of anti-

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gens in the gut. Thus, prebiotics are important in promoting the host health.

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Prebiotics have different mechanism of action on immune response. Indirect impact of

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prebiotics on immune system by affecting bacteria involved in immunomodulatory activities of

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immune system and responsible for the production of different metabolites such as SCFA having

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link with immune modulation [23]. The escalation of probiotics bacteria is due to the

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fermentation of prebiotics, which modulate the immune response in human and aquaculture, is

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an indirect effect of prebiotics on immune response [1, 24, 25]. These probiotics bacteria

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playing a significant role such as nutrient utilization, prevent infections and development of the

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immune system [26]. Compounds produced by the digestion of bacteria are acknowledged to

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influence the immune system by linking to the G-protein-coupled receptors of immune cells

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inside gut- connected lymphoid tissues [27].

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SCFAs for example butyrate and sodium phenylacetate ensure anti-inflammatory

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influences in IFN-γ-stimulated RAW 264.7 cells. They restrained the manifestation of iNOS,

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TNF-α, and IL-6 induced by IFN-γ, however they improved the expression of the anti-

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inflammatory cytokine, IL-10 [28]. These SCFA produced from prebiotics in colon by specific

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microbes having significant effect on immune system [5, 12]. The gut epithelial cell release some

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antimicrobial protein that acts as natural antibiotics belongs to the proteins families that directly

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kills the harmful microbes by the action of cell wall [21]. These epithelial cells are promoted by

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prebiotics to produce a high amount of antimicrobial protein.

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Modulations in immune system by prebiotics have been observed in all age groups and all kind

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of animal from childhood to elder age [1, 29, 30]. Prebiotics especially GOS and FOS are found

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to be beneficial in immune balance, digestive enzyme activity, beneficial gut microbiota[31], and

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reduce the risk of infectious diseases in early age group [32, 33]. However, there exist a number

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of studies on prebiotics and their impact on immune system, the objective of current review is to

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sum up the findings of presented researches that are associated with prebiotics and immune

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system by focusing their direct or indirect effect on different components of immune system.

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2. Mechanism of prebiotics action on immune system

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The digestive track is known to be home for microbiota and most of the immune system is

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associated with GIT [34, 35]. Ingested food/feed effects, evaluate the numbers of microbes and

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overall health of host was reviewed by [13, 36]. Consequently, there is an interaction between

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food, feed, microbes, and gut-associated lymphoid tissue (GALT). In order to maintain a healthy

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affiliation between microbiota and immune system, it is necessary to incorporate the beneficial

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microbes in a food and feed or provide those substance/foods that support the growth of

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beneficial microbes [1, 37]. These supporting substances are mostly prebiotics that influence

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directly on the immune system or through a series of (indirect) mechanisms that are helpful to

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retain a balanced immune system [25]. Thus, prebiotics can have a dual effect on

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immunomodulatory activities of immune system by either direct or indirect linkage. Therefore,

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we will discuss both mechanisms of actions that develop a healthy immune system.

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2.1.Direct effect of prebiotics

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Prebiotics approach to different component of immune system to balance the health of host.

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They modulate the immune response via promoting several components of the gut immune

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system.

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2.1.1. Prebiotics and mucosal immunity

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Mucosal immunity is line of defense in a region where external infectious microbes may get

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entry into the body. It consists of mucosal lymphoid tissues that secure the body against infection

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[38]. Most important part of mucosal associated lymphoid tissue (MALT) is GALT. The growth

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and immunological maturity of MALT and GALT are dependent upon provision of gut-delivered

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antigens and intestinal microbial composition [39]. Since the intestine is a first line of interaction

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between foreign objects, foods and immune system, it is very important to secure a healthy

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immune response for a better health approach. Mucosal barrier property that prevent the

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adhesion of pathogen on mucus layer; is essential for protection of gut microbes as well as host

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health, as the failure of barrier property results in GIT diseases and dysfunctioning of the

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immune system. Prebiotics and probiotics are effective weapons by various methods such as

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alteration of mucus production, decline of bacterial linkage with epithelial barrier, enrichment of

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rigid junction and increase in number of cell endurance and initiation of IgA (immunoglobulin

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[40, 41]. These influences can be achieved by direct or indirect effect by changing of tight

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junction proteins across the membrane [42, 43] and selectivity of rigid junctions, respectively

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[44]. Toll-like receptors (TLRs) are a category of proteins that play a significant role in the

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innate immune system. Activation of TLR2 by gut microbes is essential to maintain the host

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health by protecting from severe injury [45]. Specific types of prebiotics like inulin and FOS

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have been reported as an immune modulating agents [46].

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Indirect effect of prebiotics is by increasing the beneficial microbes that produce

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advantageous metabolites e.g. SCFA having anti-inflammatory properties [47, 48]. SCFAs are

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produced from prebiotics through a series of multidisciplinary fermentation steps. These are

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produced in concentrations of 70-140 mmol/L and 20-70 mmol/L in proximal and distal colon,

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respectively [49]. These have been found to stimulate the intestinal mucosal immune system

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[50]. In another study on inulin and FOS, the impact of prebiotics on the immune system has

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been clearly summarized by significant evidences [51]. It has been concluded that intake of

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inulin and fructooligosaccharides have positive impact on immune parameters and on GALT

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with special reference to cells linked payer’s patches [51]. A bunch of cases are reported to

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investigate the improvement in mucus immune response in fish. A blend of prebiotics including

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FOS, GOS and inulin were fed to common carp (Cyprinus carpio) to investigate the effects of

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prebiotics with special focus on skin mucus immune parameters [5]. The results of that

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investigation revealed that there was significant increase in skin mucus lysozyme activities due

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to GOS-fed group, while no significant difference was observed in skin mucus protease in all fed

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groups. Administration of 1-2% GOS to Caspian white fish (Rutilus frisii kutum) fry for 8 weeks

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elevated total immunoglobulin (Ig) level, lysozyme activity and improved the growth

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performance [52]. Dietary sodium propionate with inclusion level (05, 1, and 2%) to zebra fish

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for 8 weeks significantly increased appetite related gene expression and mucus gene expression

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[53]. These studies suggest that prebiotics assist in mucosal immunity by indirect is through

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production of SCFA. However, further studies on initiation of total IgA by prebiotics for fish

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cases are required to fully understand and immune modulation effect.

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2.1.2. Prebiotics and cytokine expression

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Prebiotics have multiple benefits on health of host including stimulation of immune system,

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increasing number of beneficial microbes and inflammatory properties. One of such impact is

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changing the expression of cytokines. Influence of prebiotics on immune system with special

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focus on cytokines expression has been reported in many studies through their direct or indirect

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impact (by producing SCFAs) [54-57]. Effect of prebiotics by specific and nonspecific means of

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immune response by interrelating with immune cell receptors as well as motivating endocytosis,

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respiratory rupture, phagocytosis and production of various cytokines and chemokines [58]. In

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this process, CD4+ (Th) lymphocytes, that secretes cytokines that may put forth a stimulatory or

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inhibitory consequence. After that, these B cells transfer through the bloodstream to other MALT

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location, where they make a distinction into plasma cells and release SIgA antibodies that are

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conveyed and discharged on the epithelial wall.

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Prebiotics increase the anti-inflammatory cytokines expression, regulate the expression of

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proinflammatory cytokines [59, 60], and appetite related genes expression [61, 62]. Trans-

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galacto oligosaccharide blend (B-GOS) supplemented to 44 adult volunteers with a trail duration

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of 10 weeks showed a substantial increase in probiotics especially lactic acid bacteria increase in

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phagocytosis, natural killer of cell action, anti-inflammatory cytokine interleukin-10 (IL-10)

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production and decrease of proinflammatory cytokines (IL-6, IL-1β) production [63]. Similarly

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in a comparative study of oligosaccharides including oligo-fructose along with inulin was

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compared with maltodextrin to evaluate their effect on cytokines. The results revealed that

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administration of oligofructose enriched with inulin(10g/d) results in a considerable decline in

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the levels of IL-6, TNF- α and plasma LPS [55]. Another study revealed the decline in levels of

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TNF-α, IL-6 and -8 while oral supplementation of germinated barely food stuff (a prebiotic).

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These results were well enough to explain when compared with control groups [64].

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Correspondingly, inclusion of Dietary short chain FOS and plant based protein meal

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repressed bactericidal activity, relative percentage of neutrophils, intestinal interleukin 10 (IL10)

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gene expression, while monocytes percentage, thrombocytes and lysozyme production was

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increased by feeding XOS [65]. Similarly, consumption of prebiotics by microbes that produce

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useful metabolites like SCFA have been reported to change appearance of gene responsible for

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production of cytokines interferon (IFN) –γ and interleukin (IL)-2 in rats [66]. Furthermore,

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some other SCFA like butyrate and sodium phenyacetate ensure anti-inflammatory properties in

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IFN-γ-stimulated RAW 264.7 cells. They repressed the manifestation of iNOS, TNF-α, and IL-6

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prompted by IFN-γ, however they improved the countenance of the anti-inflammatory cytokine,

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IL-10 [28]. Moreover, the study [67] conducted on hybrid tilapia (Oreochromis niloticus♀×

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Oreochromis aureus♂) to investigate the effect of shell chitin as a feed with regards to intestine

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immune status, protection against pathogenic bacteria and disease resistant. The administration

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level of 0.8% and 0.6% shell chitin reduced inflammatory response and aggregated mortality of

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tilapia after contest with A. hydrophila. Thus, these finding confirm that prebiotics are capable of

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affecting cytokines expression by increasing natural killer cells and inflammatory properties.

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2.1.3. Epithelial barrier function and prebiotics

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The intestinal zone is a multifaceted ecosystem that links inhabitant microbes and the cells of

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diverse phenotypes along with composite metabolic actions to form the epithelial wall [68]. The

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epithelial wall is exposed to a broad variety of microbes; ingested microbes get access to

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epithelial wall and contact to beneficial microbes. Association of immune cells and beneficial

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microbiota in gastrointestinal track needs a barrier and authoritarian mechanism that protect

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microbes and tissue interaction [69]. This barrier in mammalian GIT is intestinal epithelium

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which comprises of solitary cell layer that prevent the host from rigorous surrounding and

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unwanted intrusion. It is a selectively permeable to some objective beneficial for the host like

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dietary components (prebiotics, starches), electrolyte and water and strong barrier against toxins

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and objects harmful to host [70]. Intestinal epithelium is not only a barrier for pathogens but also

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offers a face covered by specific cells that produce mucus, antimicrobial peptides (AMPs), and

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antibacterial compounds for example lysozyme, which in concert with tenant microbiota present

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the first line of resistance in opposition to pathogenic microorganisms [71]. Barrier function of

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epithelium is sustained by formation of complex protein-protein network with the aim to

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mechanically attach neighboring cells and to minimize spaces between cells [69]. This complex

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protein network interlinks the epithelial cell by forming adhesive complex. These complexes

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consist of trans-membrane protein that interlink outside the cell linked together and inner side of

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the cell by adopter protein which connect the cytoskeleton. The complex protein networks

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linking epithelial cells form three strengthening complexes: desmosomes, adherence junctions

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and tight junctions (Fig. 1) [72]. These multiplexes consist of transmembrane proteins that

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intermingle extracellularly with adjoining cells and intracellularly with connecter proteins that tie

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to the cytoskeleton.

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The gut immune system devotes remarkable efforts to retain its immunological tolerance by

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severely sequestering the microbes on the epithelial wall [17]. The microbes’ distribution along

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with the mucus layer is such that only outer wall is layered with the microbes, while the inner

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layer impermeable to the penetration of bacterial considered as sterile zone of mucus making a

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defensive wall against infection [73].

Influence of prebiotics on gut immune reaction has been perceived by stimulating

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intestinal epithelial cells. GOS exert an antimicrobial effect, as it can stick to the binding site of

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bacteria on the enterocyte surface that block the adhesion of harmful microbes (Fig. 1) [74, 75].

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Human milk contains GOS, which behaves as a receptor analogue to prevent sticking together of

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harmful bacteria on the epithelial surface as shown in (Fig. 1) [76]. Thus, GOS interrelates in a

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straight line with immune system. Increment in beneficial microbiota by means of prebiotics

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encourages the discrimination and abundance of epithelial cells that regulate the intestinal

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equilibrium [77]. Some dietary fibers also have an effect on epithelial permeability, prebiotics

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and other dietary nutrients passed from membrane that facilitate the cell to perform different

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actions against pathogens and foreign bodies (Fig. 1). Study on multi-dimensional nutritious

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supplement including prebiotics showed that prebiotics stop interruption of the intestinal

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epithelial barrier function that had been damaged by tumor promoters [78]. A combination of

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prebiotics β-galactomannan (βGM) and probiotics reduce the adhesion of salmonella with

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epithelial cell of intestinal ileum and stimulate the monocyte-derived dendritic cells (Fig. 1). The

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consequence of that study was that βGM was a key factor while compared with probiotics which

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stimulated epithelial barrier, granulocyte-macrophage colony-stimulation factor, chemokines

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CCL2, CXCL8, CCL20, at protein levels (IL-6 and CXCL8) and interleukin-1α [IL-1α] IL-6

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[79]. Furthermore the direct effect of prebiotics include binding the pathogens to stop their

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pathogenic activities, helps in production of antimicrobial compounds resulting less adhesion on

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the mucus layer (Fig. 2) [22]. Supplement of mannose through type 1 fimbriae that have a finger

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type outcrop binds the salmonella results in inhibition of salmonella infection [64].

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Besides, SCFA produced from prebiotics increase the epithelial barrier function along with

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different segments of intestine indicating the significance of SCFA in intestine. These SCFA

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make physiological changes all over the intestinal tract (Fig. 2) [80-82]. Study conducted on

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juvenile golden pompano (Trachinotus ovatus) by feeding dandelion extracts for 8 weeks

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enhanced intestinal digestion and absorption by increasing muscle thickness, villus length, villus

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width and villus number in the foregut and hindgut; as well as villus number, villus width and

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muscle thickness in the midgut [83]. In addition to, the significant reduction of phylum

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Proteobacteria (phylum of gram-negative bacteria containing pathogen) was observed in

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zebrafish (Danio rerio) when chitosan silver nanocomposites were fed for 30 to 60 days

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compared with control having no chitin [84]. Thus prebiotics have a significant role to promote

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epithelial cell, prevent adhesion of pathogens on mucus layer, and help to produce antimicrobial

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compounds and direct impact on immune system. Hence, supplementation of prebiotics in diet is

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an essential strategy to enhance the epithelial barrier property for an optimistic immune response.

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2.2. Indirect action of prebiotics on immune system

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Dual response of prebiotics in term of immunomodulatory activities can be observed by

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producing useful metabolites through fermentation process or by increasing population of

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beneficial microbes in the gut [11, 85]. Gut microbiota is largely distributed into various species

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and even distributed among the various phyla, Bacterium phylum considered to be the most

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dominant phylum in the gut [13], having multiple characteristics e.g. ability to digest

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carbohydrates (fermentation or hydrolysis) and compete with pathogen for nutrition and space in

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the gut [86]. Pathogenic species e.g. E.coli and Anaerorhabdus furcosa are less capable to

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increase their growth as compared to those bacteria that modulate the immune system in the

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presence of dietary chitin fed to Atlantic cod (Gadus morhua L.) [87]. Consequently, reduce the

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growth of pathogenic microbiota results in less requirement of the immune system against the

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pathogen present in the gut. Indirect impact of prebiotics may be in different forms and involve

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multi factor mechanism to suppress the pathogenies related to immune system.

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2.2.1. Prebiotics and probiotics

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The intestinal track harbors extremely dense microbial communities, influencing the health

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of host by different metabolic activities such as digestion of food in different parts of the gastro

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intestinal tract. Elie Metchnikoff proposed the inkling that microbes present in the GIT might

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play a vital role in maintaining host health a hundred years ago [88]. Gut microbes involved in

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maintaining healthy immune system has been comprehensively characterized. Thus, there exists

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a strong relationship between gut microbes and immune system, permitting the host to bear the

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excessive quantity of antigen present in the gut [89]. Gut ecosystem alter throughout the life

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during the systematic changes of host from infancy to maturity and until the old age, and in reply

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to several factors for example foodstuff, drug and exposure to environment [90]. Evidences have

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disclosed that microbes are key concern between health and disease, gut microbial imbalance

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may leads to multiple diseases [13]. To maintain the balance of healthy microbes in gut to

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sustain host health, it is necessary to feed the microorganism in diet or to provide the supportive

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material (prebiotics) to stimulate the growth of advantageous microbes [91].

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The key substrates for microbes present in gut are prebiotics and dietary carbohydrates that

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remained unchanged in the first phase of digestion [92]. Combination of prebiotics and

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probiotics called synbiotic supplied to host confers the significant effect on immune system [93-

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95]. Different prebiotics have been found to be distinguished material for the enhancement of

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microbiota assisting in immune system [96]. Inulin found to be most known prebiotics having

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multi advantages towards developing immune response

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immunity [97] and alteration of gut microbiota and enhancement of probiotics specially

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Bifidobacteria [98]. Fructose-oligosaccharide also increases the Bifidobacteria and helpful in

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secretion of anti-flammatory material (Fig. 2) [99]. GOS a classified prebiotic is found to be

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most effective prebiotic regarding to the role of assisting immune system by directly and

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indirectly influencing and improving the health of host as well as in fish studies [5, 100]. To

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support the growth of probiotics like bifibacteria in all age group humans as well as patients of

especially its influence on innate

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irritable bowel syndrome, GOS mixture has been classified as a vital key in many studies to

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improve the immune responses [63, 101]. Another study that reveals the immune enhancing

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effect of B-GOS that increases number of beneficial bacteria, upsurge in fecal secretory IgA and

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reduction in fecal calprotectin in overweight host [102]. These bifido bacteria and lactobacilli

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probiotics have a major impact on mucosal immune system and health of host [103]. Supplement

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of prebiotics in term of infant’s formula containing mixture of galactose and fructose

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oligosaccharides results in intensification of probiotics assisting the immune response and stool

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size [33]. Similarly, the synbiotics formulation containing short chain FOS and lactobacilli were

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fed to juvenile hybrid tilapia significantly increased in beneficial microbes, resistant against

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disease and protection of fish against A. hydrophila [95]. In another study conducted by [104],

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feeding rainbow trout on synbiotics (GOS + P. acidilactici) increased antioxidant activity and

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resistance against Streptococcus iniae when compared with control. Supplementation of XOS

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(2%) to Oscar (Astronotus ocellatus Agassiz, 1831) increased the intestinal heterotrophic

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bacteria when fed for 8 weeks but intestinal histomorphology was not changed [105]. In addition

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to, the feeding of chitin to Atlantic cod (Gadus morhua L.) at level of 5% indicated that dietary

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chitin inhibited the evolution of pathogens such as E.coli and Anaerorhabdus furcosa, also

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modulate the intestinal bacterial community [87]. Altogether there exist a significant number of

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beneficial microbes in a gut, but still there is need to increase beneficial microbes by providing

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their substrate (prebiotics) to maintain a balance immune system.

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2.2.2. Prebiotics and innate immunity

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Innate immune system behaves as base line of protection by preventing the doorway of

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contagious microbes or by eradicating invaded pathogens. Attack of pathogenic microbes is

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firstly detected by innate immune system by using several pattern recognition receptors (PRRs).

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Immune system identifies special feature of pathogens (pathogen-associated molecular pattern)

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on which they are distinguished from beneficial microbes [106]. It encompasses multi barriers

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such as mucous membranes or skin with cells like e.g. T- and B-phagocytes or natural killer

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cells. It also contains soluble supporting substance medium like complementary protein or

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cytokines. Human intestine is crucial part of immune system consist multifaceted cellular

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network, produce proteins, peptides and promote host defenses. Role of innate immunity in gut

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immune defense against invading pathogen is very of the essence [100]. Different prebiotics has

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significant effects on innate immunity of human and fish [57, 107, 108]. Supplementation of

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different prebiotics including GOS to Common carp (Cyprinus carpio) increased innate

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immunity, total Ig and significant level of lysozyme [5]. In addition to, the Supplemented of

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oligo-fructose to elderly adult for 3 weeks’ results in decline of monocyte and granulocyte

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phagocytosis of pathogen microbe E. coli was recorded [109], But no control group was added in

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study to differentiate the results. Therefore, it is very difficult to interpret these effects. Effects of

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prebiotics on phagocytes cells of fish and activation of innate immunity, TLR and phagocytic

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activation have been reported [19]. Fructose polymers especially inulin-type fructan along with

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phenolic compounds for a synergistic effect act as signals in animals, promoting immune cell

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activity through (TLR) facilitated signaling, downstream signaling, disease prevention and

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immune modulator [110]. Similarly, supplementation of commercially available prebiotics

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(mixture of glucan and mannan) to juvenile pacu (Piaractus mesopotamicus) for 30 days with

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feed rate of 0-0.8% were sufficient to increase leukocytes and lysozyme activity, the number of

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thrombocytes, neutrophils and monocytes in the blood after a stressful handling, bacterial

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challenge, and minimized stress response [111]. Moreover, the addition of prebiotics in the diets

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of European seabass (Dicentrarchus labrax) increased the lysozyme activity and systematic

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innate immune response while compared with diet containing plant protein [65]. These reports

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shed light on immune modulent effect of prebiotic with special focus on innate immunity but

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further studies will be required to demonstrate the involvement mechanism of prebiotics on

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immune response.

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3. Summary and perspective

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Overall impacts of prebiotics on the immune system are well documented. Although, the

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literature exists to support that immune components like mucosal immunity, cytokines

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expression, epithelial barrier, increased beneficial microbiota and effect on innate immunity are

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promoted by consumption of prebiotics, yet there is need to investigate the mechanism of action

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of prebiotics to modulate the immune system especially the direct impact on immune system.

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The bibliographical data do not enable any clear conclusion regarding direct effect of prebiotics

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on immune parameters. There is need to conduct more studies on mucus immune parameters in

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aquaculture. Studies have shown that production of SCFAs from prebiotics by commensal

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bacteria has an impact on anti-inflammatory properties and cytokines expression. Escalation of

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probiotics bacteria by using prebiotics as a substrate confirms the importance of prebiotics in

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modulating the immune response. Numerous carbohydrates are classified as a prebiotics but the

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most studied molecules classified as immunomodulants are inulin, FOS, GOS and their

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combinations. Prebiotics have a direct influence on the epithelial barrier by promoting epithelial

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cell, prevent adhesion of pathogen on mucus and directly bind the pathogens by blocking their

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active site. At early stage after birth, prebiotics are helpful in increasing beneficial microbiota in

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the gut. However, gut ecosystem variation can be observed throughout the life that is key

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strategy to promote the immune response mechanism of the host. Similarly, probiotics are

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motivated by prebiotics to enhance the immune system activities by producing antimicrobial

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metabolites e.g. peptides, SIgA and SCFA. Furthermore, prebiotics increase only probiotics in

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the gut due to their ability to digest them instead of pathogen that lack some sacchrolytic

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enzyme.

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Even though numerous strategies employed by prebiotics and probiotics to evade the

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immune response have been identified, there are many question that need to be answered in

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respect to prebiotics mechanism of immune reaction. How prebiotics prevent adhesion of

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pathogen on mucus layer? How far is the Impact of prebiotics on immune system? At present,

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the combine studies of prebiotics and probiotics with ideal dose and duration are limited. The

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ideal dosages of synbiotics and administration regimens need to be determined in aquaculture.

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Much remains to be attained before gut immune disarrays can be prevented or treated by means

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of well-characterized probiotic strains, specific oligosaccharide prebiotics, or a combination of

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these. In vivo study of prebiotics to justify its impact on immune system will facilitate focus on

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development of novel effective therapeutic approach in future.

Fig. 1. Direct impact of prebiotics on immune system. Prebiotics modulate the immune response by preventing adhesion of pathogen on mucus, thus reducing the pathogens to invade in mucosal barrier. These modulatory molecules also bind the active site of bacteria to reduce their activity. Prebiotics are helpful in initiation of Dendritic Cell, increase permeability; interlink the epithelial cell through a tight junction that reduces the penetration of pathogens across the epithelial barrier

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prebiotics and increase the beneficial microbiota to produce useful metabolites such as SCFA that give energy to promote epithelial cell. Pathogens such as salmonella and shigella are unable to digest the prebiotics due to lack of glycoside hydrolases and saccharolytic enzyme compared with other gut microbes thus creating a less possibility and survival for pathogens. These microbes and epithelial cell produce some antimicrobial peptides that kill the pathogenic microbes causing pathogenies

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Fig. 2. Indirect impact of prebiotics on immune system. Gut microbes especially probiotics digest the

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Acknowledgement A. Nawaz is supported by China scholarship council. The authors are

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grateful to Dr. Arnie W. Hydamaka (senior instructor at University of Manitoba, Canada) for his

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support and assistance in grammar revision.

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Compliance with Ethical Standards

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Conflict of Interest; all authors declare that they have no conflict of interests.

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References

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1. Guerreiro I, Oliva-Teles A, Enes P. Prebiotics as functional ingredients: focus on Mediterranean fish aquaculture. Reviews in Aquaculture. 2017. 2. Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. The Journal of nutrition. 1995 125:1401. 3. Bindels LB, Delzenne NM, Cani PD, Walter J. Towards a more comprehensive concept for prebiotics. Nature reviews Gastroenterology & hepatology. 2015 12:303-10. 4. Liu W, Yang Y, Zhang J, Gatlin DM, Ringø E, Zhou Z. Effects of dietary microencapsulated sodium butyrate on growth, intestinal mucosal morphology, immune response and adhesive bacteria in juvenile common carp (Cyprinus carpio) pre-fed with or without oxidised oil. British Journal of Nutrition. 2014 112:15-29. 5. Hoseinifar SH, Ahmadi A, Raeisi M, Hoseini SM, Khalili M, Behnampour N. Comparative study on immunomodulatory and growth enhancing effects of three prebiotics (galactooligosaccharide, fructooligosaccharide and inulin) in common carp (Cyprinus carpio). Aquaculture research. 2017 48:3298-307. 6. Hoseinifar SH, Esteban MÁ, Cuesta A, Sun Y-Z. Prebiotics and fish immune response: a review of current knowledge and future perspectives. Reviews in Fisheries Science & Aquaculture. 2015 23:31528. 7. Carbone D, Faggio C. Importance of prebiotics in aquaculture as immunostimulants. Effects on immune system of Sparus aurata and Dicentrarchus labrax. Fish & shellfish immunology. 2016 54:172-8. 8. Wilson B, Whelan K. Prebiotic inulin-type fructans and galacto-oligosaccharides: definition, specificity, function, and application in gastrointestinal disorders. Journal of gastroenterology and hepatology. 2017 32:64-8. 9. Flint HJ, Duncan SH, Louis P. The impact of nutrition on intestinal bacterial communities. Current Opinion in Microbiology. 2017 38:59-65. 10. Louis P, O'byrne CP. Life in the gut: microbial responses to stress in the gastrointestinal tract. Science progress. 2010 93:7-36. 11. Kamada N, Chen GY, Inohara N, Núñez G. Control of pathogens and pathobionts by the gut microbiota. Nature immunology. 2013 14:685-90. 12. Fernández J, Redondo-Blanco S, Gutiérrez-del-Río I, Miguélez EM, Villar CJ, Lombó F. Colon microbiota fermentation of dietary prebiotics towards short-chain fatty acids and their roles as antiinflammatory and antitumour agents: A review. Journal of Functional Foods. 2016 25:511-22. 13. Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI. Worlds within worlds: evolution of the vertebrate gut microbiota. Nature reviews Microbiology. 2008 6:776. 14. Vandenplas Y, Zakharova I, Dmitrieva Y. Oligosaccharides in infant formula: more evidence to validate the role of prebiotics. British Journal of Nutrition. 2015 113:1339-44. 15. Reid G, Kumar H, Khan A, Rautava S, Tobin J, Salminen S. The case in favour of probiotics before, during and after pregnancy: insights from the first 1,500 days. Beneficial microbes. 2016 7:353-62. 16. Laitinen K, Poussa T, Isolauri E. Probiotics and dietary counselling contribute to glucose regulation during and after pregnancy: a randomised controlled trial. British journal of nutrition. 2008 101:1679-87. 17. Wu RY, Jeffrey MP, Johnson-Henry KC, Green-Johnson JM, Sherman PM. Impact of prebiotics, probiotics, and gut derived metabolites on host immunity. LymphoSign Journal. 2016 4:1-24. 18. Vos AP, Haarman M, VanGinkel JWH, Knol J, Garssen J, Stahl B, et al. Dietary supplementation of neutral and acidic oligosaccharides enhances Th1-dependent vaccination responses in mice. Pediatric allergy and immunology. 2007 18:304-12.

AC C

EP

TE D

M AN U

SC

RI PT

419

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

19. Song SK, Beck BR, Kim D, Park J, Kim J, Kim HD, et al. Prebiotics as immunostimulants in aquaculture: a review. Fish & shellfish immunology. 2014 40:40-8. 20. Cani PD, Everard A. Talking microbes: when gut bacteria interact with diet and host organs. Molecular nutrition & food research. 2016 60:58-66. 21. Mukherjee S, Vaishnava S, Hooper L. Multi-layered regulation of intestinal antimicrobial defense. Cellular and molecular life sciences. 2008 65:3019-27. 22. Brink M, Todorov S, Martin J, Senekal M, Dicks L. The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus. Journal of Applied Microbiology. 2006 100:813-20. 23. Hoseinifar S, Mirvaghefi A, Amoozegar M, Merrifield D, Ringø E. In vitro selection of a synbiotic and in vivo evaluation on intestinal microbiota, performance and physiological response of rainbow trout (Oncorhynchus mykiss) fingerlings. Aquaculture Nutrition. 2017 23:111-8. 24. McLoughlin RF, Berthon BS, Jensen ME, Baines KJ, Wood LG. Short-chain fatty acids, prebiotics, synbiotics, and systemic inflammation: a systematic review and meta-analysis. The American Journal of Clinical Nutrition. 2017:ajcn156265. 25. Fransen F, Sahasrabudhe NM, Elderman M, Bosveld M, El Aidy S, Hugenholtz F, et al. β2→ 1Fructans Modulate the Immune System In Vivo in a Microbiota-Dependent and-Independent Fashion. Frontiers in immunology. 2017 8. 26. Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell. 2014 157:12141. 27. Brown AJ, Goldsworthy SM, Barnes AA, Eilert MM, Tcheang L, Daniels D, et al. The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. Journal of Biological Chemistry. 2003 278:11312-9. 28. Park J-S, Lee E-J, Lee J-C, Kim W-K, Kim H-S. Anti-inflammatory effects of short chain fatty acids in IFN-γ-stimulated RAW 264.7 murine macrophage cells: Involvement of NF-κB and ERK signaling pathways. International immunopharmacology. 2007 7:70-7. 29. Firmansyah A, Dwipoerwantoro PG, Kadim M, Alatas S, Conus N, Lestarina L, et al. Improved growth of toddlers fed a milk containing synbiotics. Asia Pacific journal of clinical nutrition. 2011 20:6976. 30. Russo F, Linsalata M, Clemente C, Chiloiro M, Orlando A, Marconi E, et al. Inulin-enriched pasta improves intestinal permeability and modifies the circulating levels of zonulin and glucagon-like peptide 2 in healthy young volunteers. Nutrition research. 2012 32:940-6. 31. Mirghaed AT, Yarahmadi P, Hosseinifar SH, Tahmasebi D, Gheisvandi N, Ghaedi A. The effects singular or combined administration of fermentable fiber and probiotic on mucosal immune parameters, digestive enzyme activity, gut microbiota and growth performance of Caspian white fish (Rutilus frisii kutum) fingerlings. Fish & Shellfish Immunology. 2018. 32. Cummings J, Christie S, Cole T. A study of fructo oligosaccharides in the prevention of travellers’ diarrhoea. Alimentary pharmacology & therapeutics. 2001 15:1139-45. 33. Moro G, Minoli I, Mosca M, Fanaro S, Jelinek J, Stahl B, et al. Dosage-related bifidogenic effects of galacto-and fructooligosaccharides in formula-fed term infants. Journal of pediatric gastroenterology and nutrition. 2002 34:291-5. 34. Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science. 2012 336:1268-73. 35. Wang AR, Ran C, Ringø E, Zhou ZG. Progress in fish gastrointestinal microbiota research. Reviews in Aquaculture. 2017. 36. Xu G, Xing W, Li T, Ma Z, Liu C, Jiang N, et al. Effects of dietary raffinose on growth, non-specific immunity, intestinal morphology and microbiome of juvenile hybrid sturgeon (Acipenser baeri Brandt♀× A. schrenckii Brandt♂). Fish & shellfish immunology. 2018 72:237-46.

AC C

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ACCEPTED MANUSCRIPT

EP

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37. Bodera P. Influence of prebiotics on the human immune system (GALT). Recent patents on inflammation & allergy drug discovery. 2008 2:149-53. 38. Perez-Lopez A, Behnsen J, Nuccio S-P, Raffatellu M. Mucosal immunity to pathogenic intestinal bacteria. Nature Reviews Immunology. 2016 16:135. 39. Veldhoen M, Brucklacher-Waldert V. Dietary influences on intestinal immunity. Nature reviews Immunology. 2012 12:696. 40. Zou HK, Hoseinifar SH, Miandare HK, Hajimoradloo A. Agaricus bisporus powder improved cutaneous mucosal and serum immune parameters and up-regulated intestinal cytokines gene expression in common carp (Cyprinus carpio) fingerlings. Fish & shellfish immunology. 2016 58:380-6. 41. Modanloo M, Soltanian S, Akhlaghi M, Hoseinifar SH. The effects of single or combined administration of galactooligosaccharide and Pediococcus acidilactici on cutaneous mucus immune parameters, humoral immune responses and immune related genes expression in common carp (Cyprinus carpio) fingerlings. Fish & shellfish immunology. 2017 70:391-7. 42. De Kivit S, Tobin MC, Forsyth CB, Keshavarzian A, Landay AL. Regulation of intestinal immune responses through TLR activation: implications for pro-and prebiotics. Frontiers in immunology. 2014 5. 43. Resta-Lenert S, Barrett K. Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC). Gut. 2003 52:988-97. 44. Garcia-Lafuente A, Antolin M, Guarner F, Crespo E, Malagelada J. Modulation of colonic barrier function by the composition of the commensal flora in the rat. Gut. 2001 48:503-7. 45. Yiu JH, Dorweiler B, Woo CW. Interaction between gut microbiota and toll-like receptor: from immunity to metabolism. Journal of Molecular Medicine. 2017 95:13-20. 46. Vogt L, Meyer D, Pullens G, Faas M, Smelt M, Venema K, et al. Immunological properties of inulin-type fructans. Critical reviews in food science and nutrition. 2015 55:414-36. 47. Hoseinifar SH, Ringø E, Shenavar Masouleh A, Esteban MÁ. Probiotic, prebiotic and synbiotic supplements in sturgeon aquaculture: a review. Reviews in Aquaculture. 2016 8:89-102. 48. Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature. 2009 461:1282. 49. Engelhardt Wv, Busche R, Gros G, Rechkemmer G. Absorption of short-chain fatty acids: mechanisms and regional differences in the large intestine. Short-Chain Fatty Acids: Metabolism and Clinical Importance. 1991:60-2. 50. Goverse G, Molenaar R, Macia L, Tan J, Erkelens MN, Konijn T, et al. Diet-derived short chain fatty acids stimulate intestinal epithelial cells to induce mucosal tolerogenic dendritic cells. The Journal of Immunology. 2017 198:2172-81. 51. Seifert S, Watzl B. Inulin and oligofructose: review of experimental data on immune modulation. The Journal of nutrition. 2007 137:2563S-7S. 52. Hoseinifar SH, Zoheiri F, Dadar M, Rufchaei R, Ringø E. Dietary galactooligosaccharide elicits positive effects on non-specific immune parameters and growth performance in Caspian white fish (Rutilus frisii kutum) fry. Fish & shellfish immunology. 2016 56:467-72. 53. Hoseinifar SH, Safari R, Dadar M. Dietary sodium propionate affects mucosal immune parameters, growth and appetite related genes expression: Insights from zebrafish model. General and comparative endocrinology. 2017 243:78-83. 54. Menzel T, Lührs H, Zirlik S, Schauber J, Kudlich T, Gerke T, et al. Butyrate inhibits leukocyte adhesion to endothelial cells via modulation of VCAM-1. Inflammatory bowel diseases. 2004 10:122-8. 55. Dehghan P, Gargari BP, Jafar-Abadi MA. Oligofructose-enriched inulin improves some inflammatory markers and metabolic endotoxemia in women with type 2 diabetes mellitus: a randomized controlled clinical trial. Nutrition. 2014 30:418-23.

AC C

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ACCEPTED MANUSCRIPT

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56. Safari R, Hoseinifar SH, Kavandi M. Modulation of antioxidant defense and immune response in zebra fish (Danio rerio) using dietary sodium propionate. Fish physiology and biochemistry. 2016 42:1733-9. 57. Yousefi S, Hoseinifar SH, Paknejad H, Hajimoradloo A. The effects of dietary supplement of galactooligosaccharide on innate immunity, immune related genes expression and growth performance in zebrafish (Danio rerio). Fish & shellfish immunology. 2018 73:192-6. 58. Di Bartolomeo F, Startek J, Van den Ende W. Prebiotics to fight diseases: reality or fiction? Phytotherapy Research. 2013 27:1457-73. 59. Shokryazdan P, Jahromi MF, Navidshad B, Liang JB. Effects of prebiotics on immune system and cytokine expression. Medical microbiology and immunology. 2017 206:1-9. 60. Hoseinifar SH, Ahmadi A, Khalili M, Raeisi M, Van Doan H, Caipang CM. The study of antioxidant enzymes and immune-related genes expression in common carp (Cyprinus carpio) fingerlings fed different prebiotics. Aquaculture Research. 2017 48:5447-54. 61. Hosseini M, Miandare HK, Hoseinifar SH, Yarahmadi P. Dietary Lactobacillus acidophilus modulated skin mucus protein profile, immune and appetite genes expression in gold fish (Carassius auratus gibelio). Fish & shellfish immunology. 2016 59:149-54. 62. Miandare HK, Farvardin S, Shabani A, Hoseinifar SH, Ramezanpour SS. The effects of galactooligosaccharide on systemic and mucosal immune response, growth performance and appetite related gene transcript in goldfish (Carassius auratus gibelio). Fish & shellfish immunology. 2016 55:47983. 63. Vulevic J, Drakoularakou A, Yaqoob P, Tzortzis G, Gibson GR. Modulation of the fecal microflora profile and immune function by a novel trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers. The American journal of clinical nutrition. 2008 88:1438-46. 64. Oyofo B, DeLoach J, Corrier D, Norman J, Ziprin R, Mollenhauer H. Prevention of Salmonella typhimurium colonization of broilers with D-mannose. Poultry science. 1989 68:1357-60. 65. Azeredo R, Machado M, Kreuz E, Wuertz S, Oliva-Teles A, Enes P, et al. The European seabass (Dicentrarchus labrax) innate immunity and gut health are modulated by dietary plant-protein inclusion and prebiotic supplementation. Fish & shellfish immunology. 2017 60:78-87. 66. Cavaglieri CR, Nishiyama A, Fernandes LC, Curi R, Miles EA, Calder PC. Differential effects of short-chain fatty acids on proliferation and production of pro-and anti-inflammatory cytokines by cultured lymphocytes. Life sciences. 2003 73:1683-90. 67. Qin C, Zhang Y, Liu W, Xu L, Yang Y, Zhou Z. Effects of chito-oligosaccharides supplementation on growth performance, intestinal cytokine expression, autochthonous gut bacteria and disease resistance in hybrid tilapia Oreochromis niloticus♀× Oreochromis aureus♂. Fish & shellfish immunology. 2014 40:267-74. 68. Elshaer D, Begun J. The role of barrier function, autophagy, and cytokines in maintaining intestinal homeostasis. Seminars in cell & developmental biology: Elsevier; 2017, p. 51-9. 69. Liévin-Le Moal V, Servin AL. The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. Clinical microbiology reviews. 2006 19:315-37. 70. Odenwald MA, Turner JR. The intestinal epithelial barrier: a therapeutic target? Nature reviews Gastroenterology & hepatology. 2017 14:9-21. 71. Ganz T. Epithelia: not just physical barriers. Proceedings of the National Academy of Sciences. 2002 99:3357-8. 72. Niessen CM. Tight junctions/adherens junctions: basic structure and function. Journal of Investigative Dermatology. 2007 127:2525-32. 73. Hansson GC, Johansson ME. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Gut microbes. 2010 1:51-4.

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ACCEPTED MANUSCRIPT

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74. Gibson G, McCartney A, Rastall R. Prebiotics and resistance to gastrointestinal infections. British Journal of Nutrition. 2005 93:S31-S4. 75. Shoaf K, Mulvey GL, Armstrong GD, Hutkins RW. Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells. Infection and immunity. 2006 74:6920-8. 76. Boehm G, Stahl B. Oligosaccharides. Functional dairy products Cambridge: Woodhead Publishers. 2003:203-43. 77. Duerr CU, Hornef MW. The mammalian intestinal epithelium as integral player in the establishment and maintenance of host–microbial homeostasis. Seminars in immunology: Elsevier; 2012, p. 25-35. 78. Commane DM, Shortt CT, Silvi S, Cresci A, Hughes RM, Rowland IR. Effects of fermentation products of pro-and prebiotics on trans-epithelial electrical resistance in an in vitro model of the colon. Nutrition and cancer. 2005 51:102-9. 79. Badia R, Zanello G, Chevaleyre C, Lizardo R, Meurens F, Martínez P, et al. Effect of Saccharomyces cerevisiae var. Boulardii and β-galactomannan oligosaccharide on porcine intestinal epithelial and dendritic cells challenged in vitro with Escherichia coli F4 (K88). Veterinary research. 2012 43:4. 80. Hoseinifar SH, Zoheiri F, Caipang CM. Dietary sodium propionate improved performance, mucosal and humoral immune responses in Caspian white fish (Rutilus frisii kutum) fry. Fish & shellfish immunology. 2016 55:523-8. 81. Hoseinifar SH, Sun YZ, Caipang CM. Short-chain fatty acids as feed supplements for sustainable aquaculture: an updated view. Aquaculture Research. 2017 48:1380-91. 82. Ichikawa H, Shineha R, Satomi S, Sakata T. Gastric or rectal instillation of short-chain fatty acids stimulates epithelial cell proliferation of small and large intestine in rats. Digestive diseases and sciences. 2002 47:1141-6. 83. Tan X, Sun Z, Zhou C, Huang Z, Tan L, Xun P, et al. Effects of dietary dandelion extract on intestinal morphology, antioxidant status, immune function and physical barrier function of juvenile golden pompano Trachinotus ovatus. Fish & shellfish immunology. 2018 73:197-206. 84. Udayangani R, Dananjaya S, Nikapitiya C, Heo G-J, Lee J, De Zoysa M. Metagenomics analysis of gut microbiota and immune modulation in zebrafish (Danio rerio) fed chitosan silver nanocomposites. Fish & shellfish immunology. 2017 66:173-84. 85. Rivière A, Selak M, Lantin D, Leroy F, De Vuyst L. Bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut. Frontiers in microbiology. 2016 7. 86. Barker DJ. The origins of the developmental origins theory. Journal of internal medicine. 2007 261:412-7. 87. Zhou Z, Karlsen Ø, He S, Olsen RE, Yao B, Ringø E. The effect of dietary chitin on the autochthonous gut bacteria of Atlantic cod (Gadus morhua L.). Aquaculture Research. 2013 44:1889900. 88. Underhill DM, Gordon S, Imhof BA, Núñez G, Bousso P. Elie Metchnikoff (1845-1916): celebrating 100 years of cellular immunology and beyond. Nature Reviews Immunology. 2016 16:651-6. 89. Brown EM, Sadarangani M, Finlay BB. The role of the immune system in governing host-microbe interactions in the intestine. Nature immunology. 2013 14:660-7. 90. Quercia S, Candela M, Giuliani C, Turroni S, Luiselli D, Rampelli S, et al. From lifetime to evolution: timescales of human gut microbiota adaptation. Frontiers in microbiology. 2014 5. 91. Azimirad M, Meshkini S, Ahmadifard N, hossein Hoseinifar S. The study of enrichment capability of adult Artemia franciscana with singular or combined administration of Pediococcus acidilactici and fructooligosaccharide. International Journal of Aquatic Biology. 2016 4:96.

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92. Gibson GR, Hutkins R, Sanders ME, Prescott SL, Reimer RA, Salminen SJ, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. 2017. 93. Hoseinifar SH, Sun YZ, Zhou Z. Prebiotics and Synbiotics. Diagnosis and Control of Diseases of Fish and Shellfish. 2017:185-8. 94. Frei R, Akdis M, O’Mahony L. Prebiotics, probiotics, synbiotics, and the immune system: experimental data and clinical evidence. Current opinion in gastroenterology. 2015 31:153-8. 95. Liu W, Wang W, Ran C, He S, Yang Y, Zhou Z. Effects of dietary scFOS and lactobacilli on survival, growth, and disease resistance of hybrid tilapia. Aquaculture. 2017 470:50-5. 96. Hoseinifar SH, Eshaghzadeh H, Vahabzadeh H, Peykaran Mana N. Modulation of growth performances, survival, digestive enzyme activities and intestinal microbiota in common carp (Cyprinus carpio) larvae using short chain fructooligosaccharide. Aquaculture research. 2016 47:3246-53. 97. Macfarlane G, Steed H, Macfarlane S. Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics. Journal of applied microbiology. 2008 104:305-44. 98. Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, Louis P. Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii. British Journal of Nutrition. 2008 101:541-50. 99. Scholtens PA, Alles MS, Bindels JG, van der Linde EG, Tolboom JJ, Knol J. Bifidogenic effects of solid weaning foods with added prebiotic oligosaccharides: a randomised controlled clinical trial. Journal of pediatric gastroenterology and nutrition. 2006 42:553-9. 100. Saavedra J, Tschernia A. Human studies with probiotics and prebiotics: clinical implications. British Journal of Nutrition. 2002 87:S241-S6. 101. Depeint F, Tzortzis G, Vulevic J, I'Anson K, Gibson GR. Prebiotic evaluation of a novel galactooligosaccharide mixture produced by the enzymatic activity of Bifidobacterium bifidum NCIMB 41171, in healthy humans: a randomized, double-blind, crossover, placebo-controlled intervention study. The American Journal of Clinical Nutrition. 2008 87:785-91. 102. Vulevic J, Juric A, Tzortzis G, Gibson GR. A mixture of trans-galactooligosaccharides reduces markers of metabolic syndrome and modulates the fecal microbiota and immune function of overweight adults. The Journal of nutrition. 2013 143:324-31. 103. Gori A, Tincati C, Rizzardini G, Torti C, Quirino T, Haarman M, et al. Early impairment of gut function and gut flora supporting a role for alteration of gastrointestinal mucosa in human immunodeficiency virus pathogenesis. Journal of clinical microbiology. 2008 46:757-8. 104. Hoseinifar SH, Hoseini SM, Bagheri D. Effects of galactooligosaccharide and Pediococcus acidilactici on antioxidant defence and disease resistance of rainbow trout, Oncorhynchus mykiss. Annals of Animal Science. 2017 17:217-27. 105. Hoseinifar S, Khalili M, Sun YZ. Intestinal histomorphology, autochthonous microbiota and growth performance of the oscar (Astronotus ocellatus Agassiz, 1831) following dietary administration of xylooligosaccharide. Journal of Applied Ichthyology. 2016 32:1137-41. 106. Iwasaki A, Medzhitov R. Control of adaptive immunity by the innate immune system. Nature immunology. 2015 16:343-53. 107. Dwivedi M, Kumar P, Laddha NC, Kemp EH. Induction of regulatory T cells: a role for probiotics and prebiotics to suppress autoimmunity. Autoimmunity reviews. 2016 15:379-92. 108. Roller M, Rechkemmer G, Watzl B. Prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis modulates intestinal immune functions in rats. The journal of Nutrition. 2004 134:153-6. 109. Guigoz Y, Rochat F, Perruisseau-Carrier G, Rochat I, Schiffrin E. Effects of oligosaccharide on the faecal flora and non-specific immune system in elderly people. Nutrition Research. 2002 22:13-25.

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110. Peshev D, Van den Ende W. Fructans: prebiotics and immunomodulators. Journal of Functional Foods. 2014 8:348-57. 111. Soares MP, Oliveira FC, Cardoso IL, Urbinati EC, de Campos CM, Hisano H. Glucan-MOS® improved growth and innate immunity in pacu stressed and experimentally infected with Aeromonas hydrophila. Fish & shellfish immunology. 2018 73:133-40.

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Prebiotics promote innate and intestinal immune system The indirect effects of prebiotics on immune response are clearer than direct effects SCFAs are main key regulator of immune response by indirect method FOS, GOS and Inulin are most useful prebiotics in modulating the immune response

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