Gastrointestinal tract: the largest organ of the immune system

The term ‘gut health’ has a very complex and comprehensive definition which includes five distinct domains: (1) a balanced diet that furnishes all required nutrients; (2) a healthy mucosal layer that maintains gut integrity; (3) effective immune responses; (4) neuroendocrine and motor functions of the gut; and (5) a stable microbial community that maintains a balanced, healthy gut environment^{(Reference Montagne, Pluske and Hampson6–Reference Jha, Fouhse and Tiwari8)}.

The GI tract of pigs and poultry represents the largest mass of immune tissue in the body, consisting of more than 70 % of total immune cells^{(Reference Celi, Verlhac and Pérez Calvo7)}. Structural and immunological barriers of the GI tract protect the gut from antigens and toxins derived from food. The physical barrier includes epithelial cells, mucus and intercellular tight junctions, whereas the immunological and chemical barrier includes gastric acid, digestive secretions (for example, proteolytic enzymes, lysozymes and antibacterial proteins) and various cytokines and chemokines that regulate chemotaxis of immune cells. Tight-junction proteins regulate intestinal permeability as they act to create a seal between two adjoining enterocytes. Disruption of barrier function, therefore, leads to a condition known as leaky gut syndrome or hyperpermeability^{(Reference Liu, Li and Neu9)}. Mucosal immunoregulation in the gut is predominantly maintained by single-layered epithelial cells because it acts as the barrier between luminal contents (containing antigens and toxins) and the lymphocyte-rich lamina propria^{(Reference Seo and Shah10)}. Epithelial cells contain pattern recognition receptors (for example, toll-like receptors; TLR) that recognise antigens and bacterial lipopolysaccharides, which may ultimately cause production of various cytokines and chemokines by local intestinal immune cells in a defensive reaction. When gut health is impaired by the presence of polysaccharides or other immunostimulatory luminal components, it elicits negative effects on feed efficiency and growth performance, ultimately causing economic losses^{(Reference Jha, Fouhse and Tiwari8)}. As such, it is imperative to elucidate how dietary components relate to stimulation of immune response within the gut in order to develop nutritional strategies to mitigate such health challenges.

Xylans and xylanases

Arabinose and xylose occur in the form of AX in feed ingredients. The β(1–4)-linked xylan forms the linear backbone of AX and arabinose gets substituted on the C(O)-2 and C(O)-3 positions, with substitution of hydrocyannamic acid derivatives on the C(O)-5 position^{(Reference Pedersen, Bunzel and Schäfer11)} (Fig. 1). Arabinose substitution on the xylan backbone (i.e. arabinose:xylose (A:X) ratio) is used to characterise the structure of AX. For example, the A:X ratio in maize is 0⋅74^{(Reference Bach Knudsen12)}, in maize DDGS (distiller's dried grain with solubles) it is 0⋅77^{(Reference Tiwari, Chen and Kim13)}, in wheat it is 0⋅53^{(Reference Jha, Bindelle and Van Kessel14)}, in wheat DDGS it is 0⋅66^{(Reference Pedersen, Dalsgaard and Knudsen15)} and in macadamia nut cake it is 0⋅88^{(Reference Tiwari and Jha16)}. Due to their structural differences, a larger percentage of AX in some ingredients like oats is insoluble, whereas it is soluble in others like rye^{(Reference Bach Knudsen, Nørskov and Bolvig17)}. The molecular weight and A:X ratio of insoluble AX are generally higher than that of soluble AX^{(Reference Saulnier, Sado and Branlard18)}.

Fig. 1. Schematic illustration of coupling (hypothetical) two arabinoxylan chains via ferulic acid (forming dimer and trimer).

Xylanases are enzymes that cleave the glycosidic linkage between xylose residues in the xylan backbone. Xylanases are categorised into six different glycosidic hydrolase (GH) families: GH5, GH7, GH8, GH10, GH11 and GH43. Each of the GH families have different substrate specificities. For example, GH10 xylanases cleave xylan molecules with higher degrees of substitution whereas GH11 xylanases cleave unsubstituted xylan. However, GH11 xylanases only cleave those xylan molecules that possess three consecutive xylose without any substitution^{(Reference Mendis, Leclerc and Simsek19)}. α-l-Arabinofuranosidase degrades the terminal non-reducing α-arabinofuranose from the AX substrate. The complete degradation of AX requires a combination of enzymes^{(Reference Mendis, Leclerc and Simsek19)} (Fig. 2).

Fig. 2. Enzymic attack on arabinoxylan structure.

The overall objective of enzyme supplementation via feed is to minimise poor gut health induced by AX and to liberate other nutrients that are entrapped in the fibre, protein and starch matrix. In addition, the liberated shorter fragments of xylo-oligosaccharides (XOS) or arabinoxylo-oligosaccharides may act as prebiotic substrates for microbial fermentation in the large intestine^{(Reference Fadel, Plunkett and Li20,Reference Chacher, Kamran and Ahsan21)} . The purpose of enzyme supplementation in the feed is not to degrade AX to individual xylose and arabinose as they provide only little energy for the animal. Moreover, most of these monomers will be excreted in the urine^{(Reference Huntley and Patience1)}.

Mannans and mannanases

Glucomannan, galactomannan, galactoglucomannan (GGM) and linear mannans are the four subfamilies of the mannan family^{(Reference de Petkowicz, Reicher and Chanzy22)}. The backbone of all mannan polysaccharides contains β(1–4)-linked mannose or a combination of mannose and glucose residues, which can be substituted with α(1–6)-linked galactose^{(Reference Chauhan, Puri and Sharma23)}. Linear mannan is a homo-polysaccharide containing less than 5 % of galactose substitution^{(Reference Moreira and Filho24)}. Galactomannans consists of β(1–4)-linked mannose units with a single α(1–6)-linked galactose attached to the chain; this form is commonly found within the endosperm of legumes. Glucomannan consists of randomly arranged mannan and glucose residues both in the backbone as well as side chains in the ratio of 3:1 with a degree of polymerisation higher than 200^{(Reference Chauhan, Puri and Sharma23)}. GGM consists of α(1–6)-linked galactose residues attached to glucose or mannose residues as a terminal branch. Partial substitution of O-acetyl groups occurs in mannosyl residues of GGM. In general, GGM consists of galactose on its side chain, which forms strong hydrogen bonds and prevents other macromolecules from aligning to each other, increasing the solubility of GGM^{(Reference Chauhan, Puri and Sharma23)}.

There are mainly three mannan-degrading enzymes: β-mannanases, β-mannosidases and β-glucosidases^{(Reference Chauhan, Puri and Sharma23)} (Fig. 3). Additional enzymes (α-galactosidases and acetyl mannan esterases) are required to remove side groups from mannans^{(Reference Shallom and Shoham25)}. Each of the enzymes act on specific sites and have their own mode of action. β-Mannanases are endohydrolases and they release β-1,4-manno-oligosaccharides^{(Reference Chauhan, Puri and Sharma23,Reference Withers26)} . The β-mannosidases are exo-acting hydrolases, and they release mannose from oligosaccharides. The β-glucosidases remove 1,4-glucopyranose units that are derived from the degradation of glucomannan and GGM. The α-galactosidases help to remove side groups (i.e. α,1–6-linked d-galactopyranosyl units) from the mannan backbone and acetyl groups in the GGM are removed by acetyl mannan esterases.

Fig. 3. Enzymic attack on galactoglucomannan.

Digestion and solubility of mannans are affected by the α(1–6) side chain on the β(1–4) mannan backbone. Most of the feedstuffs used in monogastric nutrition are rich in linear mannans or galactomannans, hence β-mannanase and α-galactosidase are the most commonly used mannan-cleaving enzymes. Activity of β-mannanase on a mannotriose substrate was found to occur at a much lower rate^{(Reference Chauhan, Puri and Sharma23)} compared with mannotetraose and mannopentaose substrates, signifying greater efficacy of β-mannanase if the mannan backbone consists of more than three contiguous mannose residues.

Dietary polysaccharides and immune modulation

Many researchers have investigated the immunomodulatory effect of dietary polysaccharides, though most of them have focused on bacterial lipopolysaccharides which contain lipid A. However, the polysaccharides present in feed ingredients provided to monogastric animals are of plant origin that do not contain lipid A but still possess immunomodulatory properties^{(Reference Mendis, Leclerc and Simsek27)}. Immune activation causes the shift of energy utilisation from growth to support immune defence pathways, ultimately inhibiting the productive performance of the animal^{(Reference Huntley, Nyachoti and Patience2)}. However, the exact mechanisms by which dietary fibrous polysaccharides influence the immune response and inflammation are not fully understood. Three possible pathways highlight the immunomodulatory potential of dietary fibrous polysaccharides fed to monogastric animals: (1) direct interaction with epithelial cells or leucocytes; (2) uptake by M-cells in the Peyer's patches; and (3) uptake by macrophages and DC. Polysaccharide uptake by M-cells results in transcytosis to immune cells and subsequent cytokine production, whereas uptake by macrophages results in transportation of the polysaccharides to immune tissues including lymph nodes, spleen and bone marrow. Several studies conducted on monoculture cell lines with polysaccharides report that the cell wall polysaccharides may directly stimulate epithelial cells and monocytes, subsequently causing immunological outcomes^{(Reference Mendis, Leclerc and Simsek19,Reference Shakouri, Iji and Mikkelsen28,Reference Feng, Flanagan and Mikkelsen29)} .

Larger polysaccharides are recognised by pattern recognition receptors (PRR) found on immune cells that survey and guard peripheral tissues; these cells stimulate the production of cytokines and proliferation of lymphocytes. The PRR are sensors of the immune system and are present on and in immune and intestinal epithelial cells. The PRR involved in the recognition of NSP are TLR and C-type lectin receptors^{(Reference Ferreira, Passos and Madureira4)}. Currently, ten TLR have been identified in monogastric animals and each accept distinct pathogen-associated molecular patterns (PAMP) derived from various pathogens. The PAMP represent unique molecular signatures that are only present on microbes and not on host cells. Certain TLR (1, 2, 4 and 6) recognise lipoproteins (for example, triacyl lipopeptides), whereas other TLR (3, 7, 8 and 9) recognise nucleic acids of microbial origin (either single- or double-stranded RNA)^{(Reference Fadel, Plunkett and Li20)}. Besides bacteria, viruses, lipids, protein and nucleic acids, TLR are also able to recognise carbohydrate moieties. For example, inulin-type fructans modulate TLR2 to enhance barrier function and prevent entry of pathogens, yet C-type lectin receptors exclusively bind to NSP (for example, dectin-1 recognising β-glucan) and tend to cause immunostimulation^{(Reference Sahasrabudhe, Schols and Faas30,Reference Brown, Taylor and Reid31)} .

Role of functional groups attached to polysaccharides

All of the NSP present in the cell wall of cereal grains are composed of monosaccharides and each monosaccharide can exist in two ring forms, namely pyranose and furanose linked by glycosidic bonds in either the α or β orientation^{(Reference Bach Knudsen12)}. As a result, these polysaccharides can form different types of three-dimensional shapes, which permits a vast array of functional groups. The presence of functional groups like acetate and/or sulfate on the cell surface is known to affect the charge and solubility of polysaccharides, with sulfate groups providing negative charges and acetyl groups forming hydrophobic pockets^{(Reference Ferreira, Passos and Madureira4)}.

In addition to influencing chemical properties of the substrates per se, acetylation, sulfation and degree of branching of fibrous polysaccharides also influence gut condition, microbial ecology and immunostimulatory potential in the host consuming those polysaccharides via the diet. A higher degree of acetylation has been found to lower the immunostimulatory effect of mannan^{(Reference Ferreira, Passos and Madureira4)}. However, partially O-acetylated AX induces proliferation of thymocytes, indicating increased immunostimulatory activity^{(Reference Singh, Banerjee and Arora45)}. Sulfation of galactans has been reported to have immunostimulating activity as sulfated and carboxymethylated functional groups of β-glucan have been proven to improve solubility by impacting inter- and intra-molecular hydrogen bonding and strengthening electrostatic repulsion ability^{(Reference Ferreira, Passos and Madureira4)}.

Branching, or the degree of substitution on the backbone of fibrous polysaccharides, influences the way a dietary substrate interacts with the GI environment and ultimately affects the immune response. A higher A:X ratio leads to increased cross-linking, making it difficult for the enzymes to act upon the substrate^{(Reference Tiwari, Chen and Kim13,Reference Pedersen, Yu and Arent46)} . Cross-linking is also associated with the presence of hydroxycinnamic acid derivatives, which are most commonly found in cereals as ferulic, coumaric and sinapic acids^{(Reference Pedersen, Bunzel and Schäfer11)}. Ferulic acid is the predominant form in most cereals, with the concentration of ferulic acid being highest in maize, moderate in wheat and lower in oats^{(Reference Bach Knudsen, Nørskov and Bolvig17)}. AX in the aleurone layer is mostly insoluble due to its high ferulic acid content and heavy esterification of AX of the aleurone layer^{(Reference Pedersen, Dalsgaard and Knudsen15)}. This means that more cross-linked complex polysaccharides are present in maize and its co-products than wheat and oats. Additionally, more than 90 % of ferulic acid occurs in the bound form whereas the free and soluble forms are found at lower concentrations. Thus, occurrence of ferulic acid in the insoluble fraction of fibre is 100-fold higher than in the soluble fraction^{(Reference Bach Knudsen, Nørskov and Bolvig17)}. As such, ferulic acid is thought to form a bridge between polysaccharide chains, and thereby influence the physiochemical properties of fibre and effectively lower the efficiency of exogenous enzymes consumed by the host.

The role of solubility, viscosity and molecular weight of polysaccharides

The solubility of fibres in the gut is dependent on the type of ingredients used during the feed formulation process. Soluble fibre is better degraded by enzymes and more easily fermented by microbes in the large intestine of pigs^{(Reference Tiwari, Chen and Kim13)}, which may relate to the concentrations of diferulates present in the respective fibrous fractions^{(Reference Bunzel, Ralph and Marita47)}. Moreover, the concentration of diferulates present in the insoluble fraction of wheat is 5–7 times higher than that present in the soluble fraction of wheat^{(Reference Pedersen, Bunzel and Schäfer11)}. Higher amounts of insoluble fibre are concurrent with higher levels of diferulates and increased cross-linking^{(Reference Pedersen, Bunzel and Schäfer11,Reference Tiwari, Chen and Kim13)} . Finally, solubilised AX has a larger fraction of reducing ends that makes AX more susceptible to Maillard reactions^{(Reference Pedersen, Dalsgaard and Knudsen15)}.

Fibre viscosity is another property that is positively associated with the molecular weight of polysaccharides present in feed ingredients. For example, the molecular weight of β-glucan is higher than that of AX^{(Reference Le Gall, Serena and Jorgensen48)} and β-glucan is far more viscous than AX when in solution^{(Reference Saulnier, Sado and Branlard18)}. However, β-glucan is also more sensitive to reduction of molecular weight during passage through the small intestine^{(Reference Bach Knudsen12)}, while AX is more resistant to degradation under the same conditions, which may help to explain the greater extent to which AX negatively influences nutrient digestibility v. β-glucan.

Presence of higher concentrations of soluble fibre is also associated with increasing the viscosity of intestinal content, as soluble fibres absorb water and increase their bulk. Inclusion of high amounts of soluble AX and mannan (>0⋅4 %) in a poultry diet would increase digesta viscosity, causing an increase in epithelial cell proliferation inhibiting nutrient digestibility^{(Reference Shastak, Ader and Feuerstein5)}. Insoluble linear mannans with lower galactose substitution (for example, palm kernel meal and soyabean mannans) do not form any viscous solution in the GI tract of monogastric animals, whereas mannans with a high degree of galactose substitution (for example, copra meal or guar meal) cause a stark increase in digesta viscosity^{(Reference Shastak, Ader and Feuerstein5,Reference Chacher, Kamran and Ahsan21)} . An increase in viscosity of luminal contents is associated with an increase in the rate of villus cell loss, thereby leading to villus atrophy^{(Reference Passos, Park and Ferket49)}, slowed gastric emptying and a reduction in the contact of nutrients with enterocytes and digestive enzymes^{(Reference Tiwari, Chen and Kim13)}. Collectively, these alterations elicit a negative impact on overall nutrient availability of the monogastric diet. Since soyabeans are used as a major source of protein in most of the feed fed to monogastric animals, the presence of mannan can be presumed in most of the feeds. However, soya mannan is mainly linear mannan (containing less than 5 % galactose); they do not play a significant role in increasing intestinal viscosity^{(Reference Shastak, Ader and Feuerstein5)}.

Long villi and shallow crypts help to increase absorption of nutrients by providing a larger surface area and lower proliferation and renewal rates of enterocytes constituting villus structures results in more efficient enterocyte maturation and enzyme production^{(Reference Chacher, Kamran and Ahsan21)}. Crypt depth is also associated with cellular replacement rates as increased turnover requires more energy and amino acids, ultimately impacting growth of monogastric animals by shifting resources away from productive processes involving saleable products (i.e. meat and eggs). In monogastric animals, approximately 20 % of all the dietary energy is used by the GI tract to maintain structures supporting digestive and absorptive processes^{(Reference Celi, Verlhac and Pérez Calvo7)}. Supplementation of moderate amounts of guar meal (5–8 %) causes a marked increase in digesta viscosity in broilers^{(Reference Shastak, Ader and Feuerstein5)}, which has been proved to increase proliferation of Escherichia coli and Clostridium within the GI tract^{(Reference Shakouri, Iji and Mikkelsen28)}. Laying hens have increased digestive capacity compared with broilers and evidence suggests that the untoward effects of dietary fibre-induced viscosity are lower in layers compared with broilers fed similar concentrations of NSP^{(Reference Shastak, Ader and Feuerstein5)}. Dietary supplementation of xylan- and mannan-degrading enzymes will serve to break down polymeric fibre structures to shorter oligomeric forms, and thereby reduce viscosity of intestinal contents, enhance gut health and improve performance of monogastric species.

Oligosaccharides of xylan and mannan

Oligosaccharides are low-molecular-weight carbohydrates and the degree of polymerisation can vary from 2 to 10; however, molecules with a degree of polymerisation of less than 20 are sometimes considered as oligosaccharides due to their prebiotic potential. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon^{(Reference Gibson and Roberfroid50)}. The XOS and mannan oligosaccharides (MOS) are newly developed functional oligosaccharides produced from xylan and mannan, respectively, by enzymic hydrolysis that have beneficial immunological and health effects. Depolymerisation of XOS and MOS into oligosaccharide configurations by use of enzymes in situ (i.e. prior to feeding to monogastric animals) is neither instantaneous nor as efficient as supplementing dietary XOS and MOS directly^{(Reference Shastak, Ader and Feuerstein5,Reference Madhukumar and Muralikrishna51)} . Supplementing XOS and MOS has beneficial effects comparable with in-feed antibiotics in reducing gut inflammation^{(Reference Mendis, Leclerc and Simsek19,Reference Chacher, Kamran and Ahsan21)} . These oligosaccharides are intermediate between simple sugars and polysaccharides and behave both as dietary fibres and prebiotics.

XOS and MOS are linked together by the β-glycosidic bond of the linear polyxylose or polymannose chains, respectively^{(Reference Morgan, Wallace and Bedford52)}. Several studies have found XOS to selectively increase the proliferation of bifidobacteria to a larger extent than fructo-oligosaccharide (FOS)^{(Reference Tuohy, Rouzaud and Bruck53)} or any other oligosaccharides^{(Reference Samanta, Senani and Kolte54)}. This ‘bifidogenic effect’ of XOS has been shown in both in vitro ^{(Reference Rycroft, Jones and Gibson55)} and in vivo studies^{(Reference Hsu, Liao and Chung56)}. The XOS and MOS with monomer units of 5 or fewer are getting more attention for their potent prebiotic and butyrogenic effect.

XOS are thermostable during pasteurisation and can be autoclaved at a lower pH as compared with fructo-oligosaccharide (FOS), which is more vulnerable to decomposition at lower pH and higher temperature^{(Reference Singh, Banerjee and Arora45)}. Manno-oligosaccharides are also heat resistant as they can tolerate a higher temperature of up to 120°C for up to 20 min^{(Reference Chacher, Kamran and Ahsan21)}. Hence, both XOS and MOS should theoretically be resistant to destructive processes involved in various feed milling operations, including pelleting and some forms of extrusion. As such, XOS and MOS not only resist salivary hydrolysis or hydrolysis by gastric and pancreatic secretions, they are also not absorbed within the small intestine^{(Reference Vaquez, Alonso and Domí Nguez57)}, hence providing a substrate for microbial fermentation in the colon of pigs and caecum of poultry^{(Reference Amaretti, Bernardi and Leonardi58)}. The difference between oligosaccharide and polysaccharides of xylan and mannan is that XOS and MOS cannot form cross-linkages and are not able to stimulate immune receptors^{(Reference Jha, Fouhse and Tiwari8)}. Thus, XOS and MOS may help to reduce immune stimulation in monogastric animals and all the associated metabolic costs involved to maintain the immune response.

Bifidogenic effect of oligosaccharides

Host and gut microbes interact with each other to influence various physiological functions of the host. Functional oligosaccharides from xylan and mannan have been used in many studies to manipulate the gut microbial ecology of monogastric animals. Numerous beneficial effects on host health have been reported by oligosaccharide-mediated changes in gut microbiota including activation of the immune system^{(Reference Ibuki, Fukui and Kanatani59,Reference Ibuki, Kovacs-Nolan and Fukui60)} , production of antimicrobial factors^{(Reference Hsu, Liao and Chung56,Reference Muñoz-Tamayo, Laroche and Walter61)} , exclusion of specific enteric pathogens and changes in gut histomorphological structure^{(Reference Mendis, Martens and Simsek62)}. Bacteroidetes and Firmicutes are the polysaccharide-degrading phyla possessing the largest set of glycoside hydrolase-encoding genes^{(Reference Pourabedin and Zhao36,Reference Ejby, Fredslund and Vujicic-Zagar63)} . On the other hand, oligosaccharides are a class of compounds that help the proliferation of beneficial microbes like bifidobacteria and Lactobacillus to improve the health of animals. The role of bifidobacteria in the utilisation of oligosaccharides is gaining more attention because of their stringent selectivity of carbohydrate substrates based on the degree of polymerisation. While the majority of other enteric bacteria predominantly ferment monosaccharides, bifidobacteria preferentially utilise disaccharides and other oligomeric forms over monomeric and polymeric forms^{(Reference Amaretti, Bernardi and Leonardi58)}.

Polymeric xylans and mannans are degraded by Bacteroides and Roseburia, whereas the oligomeric form is only degraded by bifidobacteria and a few species of Firmicutes, including Lactobacillus brevis ^{(Reference Ejby, Fredslund and Vujicic-Zagar63,Reference Mirande, Kadlecikova and Matulova64)} . No other Lactobacillus sp. are capable of degrading oligomeric forms of xylan as a sole carbon source^{(Reference Moura, Barata and Carvalheiro65)}. As an exception, L. brevis can only degrade unsubstituted XOS^{(Reference Moura, Cabanas and Lourenço66)}. Uptake of oligomers of AX is mediated by the ATP-binding cassette transport system, and the only known gut microbes capable of producing this transport system is bifidobacteria^{(Reference Ejby, Fredslund and Vujicic-Zagar63)}. Most of the Bifidobacterium sp. are shown to degrade AX, but there is variation in their degradation capabilities. Bifidobacterium adolescentis and B. vulgatus can completely degrade AX^{(Reference Van Laere, Hartemink and Bosveld67)}, because they are capable of degrading both substituted and unsubstituted arabinose^{(Reference Falck, Precha-Atsawanan and Grey68)}. While B. longum and B. ovatus can partially degrade AX, B. breve and B. infantis cannot degrade XOS at all^{(Reference Van Laere, Hartemink and Bosveld67)}. The xylan-degrading enzymes produced by different Bifidobacterium sp. differ in their specificity and efficiency, though they have highly conserved solute-binding proteins that enable the degradation of AX oligomers^{(Reference Ejby, Fredslund and Vujicic-Zagar63)}. This may partly explain the variation in degradation of AX polymers exhibited by different Bifidobacterium sp.

Bacteroides can only degrade those oligomers which are larger than xylo- and manno-pentaose^{(Reference Mirande, Kadlecikova and Matulova64)}. Higher bifidogenic effects may be achieved when feeding XOS with higher arabinose substitution when compared with Bacteroides ^{(Reference Mendis, Leclerc and Simsek19)}. Bacteroides xylanisolvens growth was hindered when xylotriose with di-substituted arabinose was used^{(Reference Amrein, Gränicher and Arrigoni69)}, indicating that arabinose-substituted XOS may adversely affect bacterial growth.

Bacteroides, Bacillus and Clostridium are the three bacterial genera which have the best ability to produce mannanase that can degrade mannans^{(Reference Dhawan and Kaur3)}. MOS is found to increase Bacteroidetes proliferation^{(Reference Teng and Kim33)} and many studies have reported Lactobacillus as the main genus that is influenced by MOS. However, among all different strains of Lactobacillus, L. salivarius is most effective against Salmonella colonisation and L. crispatus against both Salmonella and E. coli ^{(Reference Teng and Kim33)}. Supplementation of 0⋅2 % dietary MOS to broilers significantly reduced profiles of E. coli in the caecum and ileum of birds^{(Reference Chacher, Kamran and Ahsan21)}. Bacterial diversity in the GI tract of monogastric animals is high, which assists in enabling various functions, including degradation of NSP that cannot be digested by host enzymes. Thus, XOS and MOS can be used as a dietary intervention to enhance the host's natural defence through modulation of gut microbiota. Supplementation of NSP-degrading enzymes can also have an impact on the microbial ecology of the gut^{(Reference Tiwari, Singh and Jha70)} that can reduce the amount of undigested substrates, degrading polymers to oligomers that potentially have functional prebiotic effect.

Oligosaccharide effects on gut barrier functions

The intestinal lining has a versatile function that helps to regulate nutrient and water absorption, while at the same time acting as a barrier excluding microbes and infectious agents that come into contact with the gut. The harmful effects elicited by pathogenic microbes occur after they attach to enterocytes of the intestinal wall and colonise the environment. However, current evidence suggests that dietary oligosaccharides may assist in clearing pathogenic microbes from the GI tract, partly through the production of mucins by goblet cells embedded among enterocytes^{(Reference Chacher, Kamran and Ahsan21)}. Mucin secretion at the brush-border membrane limits the number of bacteria that reach the epithelial layer and influence the metabolic activity of bacteria. Increased mucin production helps to improve gut barrier function as pathogenic microbes cannot penetrate the dense mucous layer^{(Reference Pourabedin and Zhao36)}. XOS have been found to increase goblet cell number and density^{(Reference Chacher, Kamran and Ahsan21,Reference Dock-Nascimento, Junqueira and de Aguilar-Nascimento71)} , which also increases mucin secretion (i.e. glycoproteins) and protein barrier factors^{(Reference Bergstrom, Guttman and Rumi72)}, thereby protecting intestinal epithelial cells. Similarly, MOS has anti-pathogenic effects, because of its ability to increase the number of sulfated goblet cells and concomitantly increase mucin secretion. Goblet cells sulfated by the use of MOS have been shown to provide greater protection for the host by enhancing pathogen degradation and reducing pathogenic microbe attachment to colonic epithelial cells^{(Reference Rajani, Dastar and Samadi73)}. Moreover, soluble mannosyl receptors secreted in mucin competitively bind to type-1 fimbriae of pathogenic microbes like Salmonella, E. coli and other Gram-negative bacteria. Thus, increased mucin production in the gut due to the presence of MOS helps in clearing pathogens by preventing microbial attachment to the intestinal wall^{(Reference Chacher, Kamran and Ahsan21)}.

Both XOS and MOS play important roles in maintaining gut integrity by increasing the secretion of IgA. Generally, mucin and secretory IgA are considered a first line of defence against the establishment of pathogenic micro-organisms in the ileum through the prevention of adhesion and subsequent epithelial invasion. Almost 98 % of all IgA-producing cells in the body reside in the small intestine^{(Reference Gutzeit, Magri and Cerutti74)}. IgA is different from any other immunoglobulin as it removes antigens/pathogens without prompting inflammatory immune responses^{(Reference Slack, Balmer and Fritz75)}. Thus, increased secretory IgA in the small intestine promotes an efficient prevention of intestinal inflammatory conditions. As such, feeding XOS via wheat bran increased the concentration of secretory IgA, which was shown to protect mucosal epithelia by preventing the attachment of pathogenic microbes^{(Reference Chen, Wang and Degroote76)}, strengthening tight junctions between epithelial cells and down-regulating the production of pro-inflammatory cytokines like TNF-α, IL-6 and interferon-γ^{(Reference Hsu, Liao and Chung56)}. Among MOS, mannobiose has been found to be the most effective at preventing bacterial infections by increasing IgA production in both pigs^{(Reference Ibuki, Fukui and Kanatani59)} and chickens^{(Reference Agunos, Ibuki and Yokomizo77)}. Supplementation of MOS increased the concentration of mucosal IgA and reduced oocyte shedding in the excreta of broilers, presumably via secretory IgA inhibiting oocyte development^{(Reference Gomez-Verduzco, Cortes-Cuevas and Lpez-Coello78)}. Additionally, supplementation of mannobiose in cultured chicken macrophage cell lines caused increased elimination of Salmonella ^{(Reference Ibuki, Kovacs-Nolan and Fukui60)}. This increase in phagocytic activity against Salmonella might be due to increased production of H₂O₂ and NO. Thus, improvement in the intestinal barrier function by the use of XOS and MOS helps prevent pathogen colonisation and enhance overall immunity and intestinal health of monogastric livestock species.