Non-Starch Polysaccharides and Their Role in Fish Nutrition

Food Chemistry 127 (2011) 1409–1426

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Food Chemistry

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Review Non-starch polysaccharides and their role in ﬁsh nutrition – A review ⇑ Amit K. Sinha a,1, Vikas Kumar b,1, Harinder P.S. Makkar b, , Gudrun De Boeck a, Klaus Becker b a Laboratory for Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium b Institute for Animal Production in the Tropics and Subtropics, University of Hohenheim, 70599 Stuttgart, Germany article info abstract

Article history: The success and sustainability of aquaculture depends on minimising the operational cost of feed that in Received 22 November 2010 general comprises 50–60% of the total cost in intensive farming. The major feed ingredient, fish meal, is Received in revised form 28 January 2011 expensive and there is increasing competition with other livestock industries for the available static sup- Accepted 8 February 2011 ply of fish meal. Hence, the incorporation of plant-derived materials in fish feeds is receiving increasing Available online 12 February 2011 attention. One of the main constraints in the utilisation of plant ingredients in aquaculture is the presence of indigestible carbohydrates, which consist primarily of non-starch polysaccharides (NSPs). These form a Keywords: part of the cell wall structure of cereals and legumes. The presence of NSPs in the diet interferes with feed Fish feed utilisation and adversely affects performance of the animal. Supplementation of NSP-degrading enzymes Non-starch polysaacharide Anti-nutritive effects in feed mitigates the adverse effects of NSPs. The effects of NSPs in pigs and poultry have been widely Nutrient metabolism studied; however little information exists for fish. This review synthesizes the available information on Immunostimulants fish and highlights the knowledge gaps. It is hoped that this review will provide a momentum to the research on the roles of NSPs in fish nutrition and physiology and on the efficient use of NSP-degrading enzymes. Ó 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 1410 2. Classification of non-starch polysaccharide ...... 1410 2.1. Cellulose...... 1411 2.2. Non-cellulosic polymers ...... 1412 2.2.1. Arabinoxylans ...... 1412 2.2.2. Mixed-linked b-glucans ...... 1412 2.2.3. Mannans ...... 1412 2.3. Pectic polysaccharides...... 1413 2.3.1. Arabinans, galactans and arabinogalactans...... 1413 3. Non-starch polysaccharides...... 1414 4. Non-starch polysaccharides in fish feed ...... 1414 5. Methods for non-starch polysaccharides quantification ...... 1415 5.1. Gravimetric analysis ...... 1415 5.2. Monomeric component analysis...... 1416 6. The anti-nutritive effect of non-starch polysaccharides ...... 1416 6.1. Modulation in digesta viscosity ...... 1416 6.2. Alteration in gastric emptying and rate of passage...... 1417 6.3. Alteration of gut physiology, gut morphology, native gut microflora and mucus layer of gut ...... 1417 7. Effect of non-starch polysaccharides on nutrient metabolism ...... 1418 7.1. Effect on glucose ...... 1418 7.2. Effect on protein ...... 1418 7.3. Effect on lipid and cholesterol level ...... 1419 7.4. Effect on minerals ...... 1419

⇑ Corresponding author. Tel.: +49 711 45923640; fax: +49 711 45923702. E-mail address: [email protected] (H.P.S. Makkar). 1 These authors contributed equally to this review.

8. Effect on growth performance and body composition...... 1419 9. Non-starch polysaccharides and effect on gelatinisation...... 1420 10. Interaction with antibiotics ...... 1420 11. Purified non-starch polysaccharides as immunostimulants ...... 1420 12. Non-starch polysaccharides degrading enzymes ...... 1422 12.1. Mechanism responsible for enzymic degradation of non-starch polysaccharides...... 1422 12.1.1. Disruption of cell wall integrity...... 1422 12.1.2. Reduction of digesta viscosity ...... 1422 12.1.3. Stimulation of bacterial population...... 1422 13. Conclusions...... 1423 References ...... 1423

1. Introduction charides are also thought to be responsible for a slower rate of gastro-intestinal passage of NSP-containing diets in fish (Storebak- The growing demand for fish and limited supply from wild cap- ken, Kvien, Shearer, Grisdale-Helland, & Helland, 1999). Feeding of ture are giving momentum to the development of aquaculture. The salmonids with diets incorporating NSP has been shown to reduce progress of culture-based fisheries is determined mainly by the the availability of nutrients (Storebakken & Austreng, 1987). quality of feed delivered. Fish meal (FM) having high protein con- At present approximately 2.0 billion tonnes of cereal grains tent and favourable amino acid profile is highly preferred by fish and 140 million metric tonnes of legumes and oil seeds are pro- culturists. The total world FM production is about 5–6 million ton- duced worldwide per year and approximately 230 million metric nes per annum, which accounts for 4–5% of total fish production of tonnes of fibrous materials are produced as a by-product. This 144 million metric tonnes (Food, 2008). In spite of being the most wide availability of plant resources can very well be utilised as important protein source in commercial feeds, production of FM is cheaper fish feed ingredients, through proper management of restricted to certain parts of the world only; as a result it is becom- the NSPs in these plant materials. Feed processing and utilisation ing too expensive for many aquaculture practising countries. Fur- of exogenous enzymes (b-glucanase and b-xylanases) have been thermore, the abundance of FM appears to be ending, since the used to decrease the negative effects of NSP and thus to improve level of FM production is expected to remain stable over the next the nutritive value of feed. Moreover, there is a contemporary 10 years (Mazurkiewicz, 2009). As a consequence, the fisheries sec- trend to seek feed ingredients which may contribute to better tor may have to undergo a recessionary phase over the coming health by interfering with colonisation and microbial growth in years. In order to provide sustainability, therefore, it is of utmost the gut. In this regards NSPs such as b-glucans and mannose have importance to reduce the presence of FM in aquafeeds and replace been shown to have immunostimulating activities (Kumar, Sau- it with plant-based sources. The higher availability and low cost of rabh, Sahu, & Pal, 2005). plant-based feeds give them advantages over FM. Although the carbohydrate component of grains and legumes may provide a cheap source of dietary energy for fish, it is poorly utilised, compared to 2. Classification of non-starch polysaccharide protein and lipid, by most fish species (Allan et al., 2000). In addition, the quality and level of protein in, and palatability of, plant- The term NSP covers a large variety of polysaccharide mole- based protein sources are generally inferior to FM. However, the cules, excluding a-glucans (starch). NSPs have been classified main limitation with plant-derived materials, such as legume based on different criteria. Historically, the classification was seeds, soybean meal, different types of oilseed cake, canola (rape- based originally on the methodology used for extraction and seed) meal, sunflower oil cake, root tuber meal, is the presence isolation of polysaccharides. The residue remaining after a series of a wide range of anti-nutritional factors, such as protease inhib- of alkaline extractions of cell wall materials was called cellulose, itors, non-digestible carbohydrates, lectins, saponins, phytates and and the fraction of this residue solubilised by alkali was named possibly allergenic storage proteins (Francis, Makkar, & Becker, hemicellulose (Neukom, 1976). Another classification was based 2001). In addition to these factors that hamper digestion in fish on the differences in solubility. This classification includes three (Refstie, Svihus, Shearer, & Storebakken, 1999; Storebakken, categories of NSP, namely crude fibre (CF), neutral detergent fi- Shearer, & Roem, 1998), non-starch polysaccharides (NSPs) play bre (NDF) and acid detergent fibre (ADF). CF refers to the an important role. In general, NSPs are a complex group, composed remnants of plant material after extraction with acid and alkali, predominantly of linked monomers of hexoses and pentoses, e.g., and includes variable portions of insoluble NSP. NDF comprises galactose, glucose, arabinose, xylose and mannose (van Barneveld, the insoluble portion of NSP plus lignin, while ADF refers to a 1999). The NSP content in wheat and lupin may account for 25% portion of insoluble NSP comprised largely, but not solely, of and 50% of the total grain and seed respectively and acts as the pri- cellulose and lignin. However, this basis of categorisation lacked mary energy storage carbohydrate in lupin (van Barneveld, 1999). precision with respect to both chemical structures and biological However, in fish and other monogastric animals enzymes such as functions and, moreover, the nutritional significance of val- b-glucanases or b-xylanases that digest NSPs are scarce or non- ues obtained using this method in monogastric nutrition is existent (Kuz’mina, 1996). Consequently, the dietary NSPs remain doubtful. indigestible and cannot be used as an energy source. The addition Bailey (1973) proposed a clearer classification of NSP into three of NSP-containing feedstuffs to the diets of monogastric animals, main groups, namely cellulose, non-cellulosic polymers and pectic for example, broiler and swine, reduces the apparent digestibility polysaccharides. Arabinoxylans, mixed-linked b-glucans, mannans, of the diet and has negative impacts on growth. However, only a and xyloglucan come under the category of non-cellulosic poly- limited number of such studies have been conducted in fish. Refstie mers while polygalacturonic acids substituted with arabinan, et al. (1999) have demonstrated negative effects of NSPs on diges- galactan and arabinogalactan are included in the group of pectic tion and absorption of lipid in Atlantic salmon. Non-starch polysac- polysaccharides (Table 1). A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1411

Table 1 Classiﬁcation of non-starch polysaccharides.

Category Monomeric residue Linkage Sources Cellulose Glucose b-(1 ? 4) Most cereals and legumes Non-cellulosic polymers Arabinoxylans Arabinose and Xylose b-(1 ? 4)-linked xylose units Wheat, rye, barley, oat, rice, sorghum Mixed-linked b- Glucose b-(1 ? 3) and b-(1 ? 4) Oat and barley glucans Mannans Mannose b-(1 ? 4) Coffee seed Galactomannans Galactose and b-(1 ? 4)-linking mannan chains with a-(1 ? 6)-linked galactosyl side groups Locust bean gum and guar gum mannans Glucomannans Glucose and mannans b-(1 ? 4)-linked mannan chain with interspersed glucose residues in the main Sugar-beet pulp, lilies, irises chain Pectic polysaccharides Arabinans Arabinose a-(1 ? 5) Cereal co-products Galactans Galactose b-(1 ? 4) Sugar bean meal, sugar-beet pulp Arabinogalactans (Type Arabinose and b-(1 ? 4) galactan backbone substituted with 5-linked and terminal arabinose Grain legumes I) Galactose residues Arabinogalactans (Type Arabinose and b-(1 ? 3,6)-linked galactose polymers associated with 3- or 5-linked arabinose Rapeseed cotyledon II) Galactose residue

or other reserve polysaccharide, should be low (Brett & Waldron, 1996). Cellulose is a straight-chain polymer where no coiling or branching occurs, and the molecule adopts an extended and rather stiff rod-like conformation, aided by the equatorial conformation of the glucose residues. The multiple hydroxyl groups on the glucose Fig. 1. Chemical structure of cellulose. residues from one chain form hydrogen bonds with oxygen mole- cules on the same or on a neighbouring chain, holding the chains firmly together side-by-side (Fig. 1). The chains can stack together to form larger microfibrils which make cellulose highly insoluble in 2.1. Cellulose water, but can swell in concentrated sodium hydroxide solutions. Through the use of hydrogen-bond breaking reagents, such as N- Cellulose is a complex polysaccharide, consisting of 3000 or methylmorpholine N-oxide, cellulose can be brought into solution. more b-(1 ? 4) linked D-glucose units. This bond generally makes Moreover, to be used as a dietary supplement, cellulose-rich maize cellulose ingestible for monogastric animals, due to lack of the en- bran can be converted to a cellulosic gel through thermal and shear zyme cellulase in the digestive tract. Cellulose is the basic struc- treatments, followed by alkaline peroxidation and shearing tural component of plant cell walls and comprises about 33% of (Fincher & Stone, 2004). Cellulose microfibrils may also associate all vegetable materials. It is the most abundant of all naturally- with water and matrix polysaccharides, such as the (1 ? 3, occurring organic compounds, comprising over 50% of all the car- 1 ? 4)-b-D-glucans, heteroxylans (arabinoxylans) and glucomann- bon in vegetation. ans (Fincher & Stone, 1986a). Cellulose quantity in whole grains can vary from species to spe- The hydrolysis of cellulose is limited in animals but can be occur cies and is largely a consequence of the thickness of the husk and in some microbes, such as bacteria and fungi (Xiao & Xu, 2002). seed coat. Cells which contain more cellulose tend to have thicker Since fish lack cellulase in their intestines, cellulose is indigestible and stronger cell walls. Seed endosperm cells have only thin cell in fish; and therefore it is of no nutritional value in formulated fish walls and in a well-filled grain the proportion of cellulose to starch, feeds. However, developments in fish nutrition and aquaculture

Fig. 2. Chemical structure of arabinoxylan. 1412 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 technology have encouraged the use of cheaper feed ingredients; Apart from covalent cross-links, arabinoxylans may also form including those containing cellulose. Therefore, it is important to ‘‘junction zones’’ by intermolecular hydrogen bonding between develop methods for the improvement of enzymatic hydrolysis of unsubstituted regions of the xylan backbone (Fincher & Stone, cellulose. 1986b). Not only can arabinoxylans establish covalent cross-links, they may also form ‘‘junction zones’’ by intermolecular hydrogen 2.2. Non-cellulosic polymers bonding between unsubstituted regions of the xylan backbone (Fincher & Stone, 1986b; Sørensen et al., 2007). Such type of inter- 2.2.1. Arabinoxylans action of arabinoxylans may be of great importance in determining Arabinoxylans have been identified in a variety of tissues of the their conformational changes and solubility properties, and thus main cereals: wheat, rye, barley, oat, rice, sorghum (Fincher & their anti-nutritional activities. Stone, 1986b). Although these polysaccharides are minor components of entire cereal grains, they constitute an important part of 2.2.2. Mixed-linked b-glucans plant cell walls. Thin walls that surround the cells in the starchy The physical and physiological properties of b-glucans are of endosperm and the aleurone layer in most cereals consist predom- commercial and nutritional importance. Increasing interest in b-glu- inantly of arabinoxylans (60–70%); exceptions are endosperm cell cans during the last two decades is largely due to their acceptance as walls of barley (20%) and rice (40%) (Fincher & Stone, 1986b). functional, bio-active ingredients (Cui & Wood, 2000). Cereal b-glu- Non-endospermic tissues of wheat, particularly the pericarp and cans have been associated with the reduction of plasma cholesterol testa, also have very high arabinoxylan content (64%) (Selvendran and a better control of postprandial serum glucose levels in humans & DuPont, 1980). and animals (Bhatty, 1999). The mixed-linked b-glucans are unique The structures of cereal arabinoxylans are composed predomi- to the Poales, the taxonomic order that includes cereal grasses. They nantlyoftwopentoses,arabinoseandxylose(Izydorczyk&Biliaderis, are also known as cereal b-glucans and are located in the subaleu- 1995). Their molecular structure consists of a linear backbone of b- rone and endospermic cell wall (Ebringerová, 2006) where they (1 ? 4)-linked xylose units to which substituents are attached associate with cellulose microfibrils during cell growth. Oats and through O-2 and O-3 atoms of the xylosyl residues (Perlin, 1951) barley contain 3–12% and more depending on the cultivar. (Fig. 2). Arabinoxylans form highly viscous aqueous solutions which The ubiquitous structural features of these polysaccharides are may create problems when wheat is used in processes, such as well established. The structural features of b-glucans are important production of grain spirits. The degree of arabinose substitutions will determinants of their physical properties and functionality, includ- influence the conformation adopted by arabinoxylansand the resulting their physiological responses when they are considered as ing viscosity of solutions. ingredients in cereal-based foods and other formulated products. When the arabinose residues are stripped off the xylan back- These features include ratios of linkages joining glucose units, bone (using oxalic acid), aggregation appears at a xylose-to-arabi- presence and amount of long cellulose-like fragments, and molec- nose ratio of about four and precipitation occurs when this is ular size (Izydorczyk & Biliaderis, 2000). In general, b-glucans con- increased above ten (Sternemalm, Höije, & Gatenholm, 2008). sists of a linear chain of glucose units joined by both b-(1 ? 3) and The loss of arabinose side chains also correlates with a loss in b-(1 ? 4) linkages (Bengtsson, Åman, & Graham, 1990)(Fig. 3). water-binding capacity (Sternemalm et al., 2008). When this is ab- It should be noted that the mixed-linked b-glucans and cellu- sent the molecule binds less water and becomes less soluble. The lose are both comprised of b-linked-glucose units but there is little degree of arabinose substitution has little influence on the overall similarity in their physical properties. Cellulose consists only of semi-flexible conformation and hence the viscosity (Sternemalm (1 ? 4)-b-linkages and is therefore stiff, highly crystalline and et al., 2008). However, in wheat flour, the distribution of the type non-soluble. On the other hand, the b-(1 ? 3)-linkages break up of substitution is not random but the distribution of substituted the regular structure of b-(1 ? 4) chains of the b-glucan molecule (irrespective of of the substitution type) residues along the chain and make it soluble and flexible (Anderson & Bridges, 1993). There- appears random (Dervilly-Pinel, Tran, & Saulnier, 2004). The arab- fore, the b-glucans are often isolated by aqueous extraction fol- inose residues may also be linked to other groups attached, such as lowed by precipitation with ammonium sulphate (McCleary, glucuronic acid residues, ferulic acid crosslinks and acetyl groups 1986). (Sørensen, Pedersen, & Meyer, 2007). The potential application of b-glucans as food hydrocolloids has Most of the arabinoxylans in cereal grains are insoluble in water been also proposed. In addition, b-glucans have been shown to because they are anchored in the cell walls by alkali-labile ester- form gels under certain conditions (Lazaridou, Biliaderis, & like cross links (Mares & Stone, 1973). But the arabinoxylans not Izydorczyk, 2003) and can be utilised as thickening agents to mod- bound to the cell walls can form highly viscous solutions and they ify the texture and appearance in gravies, salad dressings and ice can absorb about ten times their weight of water. In the presence cream formulations (Wood, 1986) or may be used as fat mimetics of oxidative agents, such as H2O2/peroxidase, arabinoxylans can in the development of calorie-reduced foods (Inglett, 1990). rapidly develop a gel network, as a result of the re-establishment of cross-links (Geissmann & Neukom, 1973). Fully cross-linked ara- 2.2.3. Mannans binoxylans hold up to 100 g of water per g polymer (Izydorczyk & Linear chains of b-(1 ? 4) mannan are found in the cell walls of Biliaderis, 1995). the coffee seed endosperm (Wolfrom, Layer, & Patin, 1961). In most

Fig. 3. Primary structure of b-D-glucans (Ebringerová, 2006). A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1413

Fig. 4. Primary structure of galactomannans (Ebringerová, 2006).

Fig. 5. Primary structure of glucomannans (Ebringerová, 2006). cases, these polysaccharides are highly insoluble in water and very chemical properties of the storage glucomannan, known as ‘konjac dense. Accordingly, it has been suggested that mannans form the mannan’ in the food industry, have been reinvestigated (Nishinari, molecular basis for the hardness of the plant. Mannan has also Williams, & Phillips, 1992). The results indicate that this polysac- been reported to be present in the cell walls of several siphona- charide, composed of glucose and mannose in the ratio 1–1.6, ceous green algae in the families Acetabularia, Codium and has the backbone slightly O-acetylated, and branched (about 8%) Halicoryne (Frei & Preston, 1968). Furthermore, mannan is also at position 6 of the glucopyranose residues. The branches termi- found in some red algae, such as Porphyra umbilicalis (Jones, nated by both glucopyranose and mannopyranose have been sug- 1950). In some of these algae, mannan is the main structural poly- gested as a new structural feature. mer and displays microﬁbrillar morphology (Chanzy, Grosrenaud, Vuong, & Mackie, 1984). The mannan-type hemicelluloses, can be divided into two 2.3. Pectic polysaccharides groups: (i) galactomannans and (ii) glucomannans (Ebringerová, 2006). Pectins consist mainly of D-galacturonic acid (GalA) units (Ridley, O’Neill, & Mohnen, 2001; Thakur, Singh, & Handa, 1997) as the backbone, joined in chains by means of a-(1 ? 4) glycosidic 2.2.3.1. Galactomannans. Galactomannans are reserve polysaccha- inkage. These uronic acids have carboxyl groups, which in the pres- rides in the seed endosperm of leguminous plants (Leguminosae). ence of divalent cations (usually calcium) have considerable effects They are water-soluble and can imbibe water, thus providing a on viscosity, solubility, and gelation formation (Thakur et al., water-holding function for the seed (Reid, 1985). They are com- 1997). posed of b-(1 ? 4)-linked mannan chains with a-(1 ? 6)-linked galactosyl side-groups (McCleary, 1985)(Fig. 4). Both the solubility and the viscosity of the galactomannans are inﬂuenced by the 2.3.1. Arabinans, galactans and arabinogalactans mannose-to-galactose ratio, which can vary from 1 to 5 (Reid, These three classes of polymers together constitute the neutral 1985). Furthermore, the distribution of the substituents can vary pectic substances. Pure arabinans and galactans are present in considerably, which also affects the physical properties of galacto- plant cell wall but in a very low amount. The arabinans are poly- mannans (Daas, Schols, & de Jongh, 2000). Two of the most well mers of (1 ? 5)-a-L-arabinose residues with some degree of characterised galactomannans are found in locust bean gum and branching through O-2, O-3 or both positions. Galactans are b-1,4 guar gum, isolated from the seeds of Ceratonia siliqua and Cyanap- linked linear polymers, which can possess a small number of 6- osis tetragonolobus, respectively (Rol, 1973). linked residues (Ghosh & Das, 1984). The arabinogalactans occur in two distinct types in plant cell 2.2.3.2. Glucomannans. Glucomannans are present as a minor com- walls. Type I, which is very common in grain legumes, is character- ponent in cereal grains (Fincher & Stone, 1986b) and act as storage ised by b-(1 ? 4) galactan backbone substituted with 5-linked and polysaccharides in the seeds of certain annual plants, for example terminal arabinose residues (Cheetham, Cheung, & Evans, 1993). some lilies (Liliaceae) and irises (Iridaceae)(Meier & Reid, 1982). The type II arabinogalactan is commonly found in rapeseed cotyle- Glucomannans are also found in the bulbs, roots and tubers of sev- don (Siddiqui & Wood, 1972). Type II is characterised by b- eral other types of plants. Many of these glucomannans are water- (1 ? 3,6)-linked galactose polymers associated with 3- or 5-linked soluble and are composed of a b-(1 ? 4)-linked mannan chain with arabinose residue. Unlike type I arabinogalactans, type II are not a interspersed glucose residues in the main chain; they are often structural component of the cell wall but are thought to be associ- acetylated (Fig. 5). The mannose-to-glucose ratio ranges from 4:1 ated with extracellular space and with plasmalemma. However, a to less than 1:1 (Meier & Reid, 1982). The structural and physico- low molecular weight type II arabinogalactan associated with a 1414 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426

Table 2 Non-starch polysaccharide content (g/kg dry matter) of ingredients used in ﬁsh feed.

Ingredients Cellulose Total NSP Lignin Soluble NSPa Insoluble NSP Cereals Maize 22 97 11 9 66 Wheat 20 119 19 25 74 Wheat bran 72 374 75 29 273 Rye 16 152 21 42 94 Barley: hulled 43 187 35 56 88 Barley: hull-less 10 124 9 50 64 Corn gluten 75 351 – 34 242 Oats: hulled 82 232 66 40 110 Oats: hull-less 14 116 32 54 49 Oat hull meal 196 505 148 13 295 Legumes/Seed meal Peas 53 180 12 52 76 Soybean meal 62 217 16 63 92 Rapeseed meal 52 220 134 55 123 Lupins 131 405 – 134 139 Sunflower cake 123 315 – 57 136 Jatropha curcas kernel meal (non-toxic genotype) 56 136b –– – Detoxified Jatropha curcas kernel meal (toxic genotypes) 65–76 160b –– – Detoxified Jatropha curcas protein isolate (toxic genotypes) – 105b –– – Miscellaneous Sugar-beet pulp 195 779 35 407 177 Cottonseed cake 92 257 – 61 103 Cottonseed meal 90 283 – 66 127 Alfalfa leaf meal 139 329 _ 77 113

a Non-cellulosic polysaccharides. b Our unpublished data. hydroxyproline-rich peptide (arabinogalactan proteins, AGPs) has hand, the enzymes required to digest NSP, such as b-glucanase also been isolated from wheat flour (Fincher & Stone, 1974). and b-xylanases, are very scarce or even absent among fish species (Kuz’mina, 1996). 3. Non-starch polysaccharides Apart from NSPs, other unavailable carbohydrates in food are oligosaccharides, fructans, lignin and resistant starch. However, Non-starch polysaccharides are complex polysaccharides other NSPs along with lignin and resistant starch are the major constitu- than the starch. These polysaccharides are typically long polymeric ents of total dietary fibre. Lignin is a high-molecular-weight poly- carbohydrate chains containing up to several hundred thousand mer composed of phenylpropane residues, formed by the monomeric units. Non-starch polysaccharides can comprise up to condensation of the aromatic alcohols, cinnamyl, guaiacyl and 90% of the cell wall of plants (Selvendran & DuPont, 1980). The syringyl alcohols (Southgate, 1993). Resistant starch can be defined most abundant plant cell wall NSPs include cellulose, hemicellu- as that fraction of dietary starch, which escapes digestion in the lose and pectins; while fructans, glucomannans and galactomann- small intestine. It is the sum of starch and degradation products ans belong to the group of NSP that is not so abundant as cellulose, of starch. hemicellulose or pectins and serves as the storage polysaccharides within the plants. Mucilages, alginates, exudate gums, b-glucans and various modified polysaccharides are other constituents of 4. Non-starch polysaccharides in fish feed the non-starch polysaccharides (Asp, Schweizer, Southgate, & Theander, 1992). The physiological impact of individual NSPs is The NSPs in aquaculture feeds are present as an integrated part dependent on the sugar residues present and nature of the linkage of the cell wall of plant ingredients and also in a purified soluble present between these residues. However, NSPs differ from starch form, such as guar gum, to stabilise the pellet. In general NSPs not only in the type of monomers present but also differ by the are present in two wide categories of crops, namely cereal grains number and type of monomeric units linked together, the order and legumes. Non-starch polysaccharides in cereal grains are com- in the chain and the types of linkages between the various mono- posed predominantly of b-glucans, arabinoxylans, and cellulose. mers. Starch is composed entirely of glucose monomers, which are Wheat, rye and triticale contain substantial amounts of both solu- linked by a-glycosidic bonds while NSPs are composed of different ble and insoluble NSP, while very low levels of NSPs are present in kinds of monomers, which are linked predominantly by b-glyco- corn and sorghum. Grain legumes also contain considerable sidic bond. The difference in bonding structure has profound ef- amounts of NSPs. Pectic polysaccharides are the main NSPs present fects on digestibility, as different classes of enzymes are required in the cotyledon of legumes. Cellulose and xylans, besides being for the hydrolysis of a- and b-glycosidic bond. The predominant the major NSPs in cereal grains, are only found in the hulls or husks starch digestive enzymes are a-amylase (1,4-a-D-glucan glucano- of most legumes. hydrolase), a-glucosidase (1,4-a-glucosidase) and oligo-1-6-gluco- Apart from cereals and legumes, NSPs are also present in other sidase. In com bination, these enzymes specifically hydrolyse the plant parts, such as roots, tubers and leaves. These ingredients are a-glycoside bonds of starch to yield glucose. The activity of these used in many countries as possible feed resources. The NSP content enzymes in fish intestine varies with fish species, and is based on of ingredients used as aqua feeds is presented in Table 2. Moreover, their feeding habits. Activity of these enzymes is high in herbivo- an extensive report on carbohydrate and lignin contents of plant rous, medium in omnivorous and low in carnivorous fish (Krog- materials used in animal feeding has already been published by dahl, Hemre, & Mommsen, 2005; Kuz’mina, 1996). On the other Bach Knudsen (1997). A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1415

5. Methods for non-starch polysaccharides quantification However, it is not an accurate estimation of total NSPs, since the recovery of cellulose, hemicelluloses and lignin is low (Van Soest It is difficult to develop an accurate analytical method for esti- & McQueen, 1973). The refined form of gravimetric crude fibre mating NSP content in feed. This is because of the complexity and analysis is the detergent method of fibre analysis, which delineates diversity of the polysaccharides involved. In general gravimetric the form of fibre present in feedstuffs. The two forms of detergent and monomeric component analytical approaches are used for fibre are neutral detergent fibre (includes cellulose, hemicellulose quantifying NSPs. The gravimetric method of fibre analysis and lignin) and acid detergent fibre (includes cellulose and lignin), assumes that all residues are present in fibre, whereas component the difference of these two fractions is an estimate of hemicellulose analysis quantifies the amount of constituent sugars present in a in a feed. Even though detergent methods of fibre analysis have substrate and then, via summation, determines the total NSP many advantages over crude fibre estimation, both underestimate concentration. the amount of total fibre in a feed, due to inability to recover pectins, mucilages, gums and b-glucans which are soluble components of fibre. Asp, Johansson, Hallmer, and Siljeström (1983) developed 5.1. Gravimetric analysis an enzyme-based gravimetric method, in which a sample is pre- treated with enzymes for the digestion of starch and protein, Gravimetric method is the traditional way of fibre analysis, followed by the recovery of soluble components via precipitation which involves chemical or enzymatic solubilisation of dietary pro- in ethanol and the insoluble components by filtration. This method tein, starch and fat, followed by weighing of the insoluble residue. was further modified by Jeraci, Lewis, Van Soest, and Robertson The crude fibre estimation is an example of gravimetric analysis. (1989), incorporating a urea enzymatic dialysis to assure the

Table 3 Factors responsible for anti-nutritive effects of non-starch polysaccharides.

Factors Effects References

Changes in digesta Reduced mixing of digestive enzymes and Choct et al. (1996); viscosity substrates Ikegami et al. (1990); Hossain et al. (2001); Hindered effective interaction of digestive Amirkolaie, Leenhouwers, Verreth, and Schrama (2005); enzyme at the intestinal mucosal surface Leenhouwers et al. (2007a, 2007b) Increased residence time of the digesta Increased intestinal volatile fatty acid (VFA) production Reduced absorption of minerals especially sodium ion Impaired nutrient digestion and absorption Reduced animal performance Alteration in the gastric Reduced rate of gastric emptying Rainbird and Low (1986); emptying and rate of Increased rate of passage of stomach content Potkins, Lawrence, and Thomlinson (1991); Shimeno et al. (1992); Kaushik et al. passage Delayed intestinal absorption of glucose (1995); Refstie et al. (1999); Bach Knudsen (2001); Hossain et al. (2001); Reduced plasma cholesterol and glucose Leenhouwers et al. (2007a, 2007b) levels Alteration of gut Hinder endogenous secretion of water, pro- Pettersson and Åman (1989); physiology teins, electrolytes and lipids Angkanaporn et al. (1994); Enhanced bile acid secretion, and significant Choct (1997); loss of these acids in the faeces Hossain et al. (2001) Hampered absorption of lipids and cholesterol in intestine Limited intestinal enzyme activity Alteration in the gut Increased size and length of digestive organs Baserga, (1985); Jin et al. (1994); Nabuurs (1998); McDonald (2001); Iji et al. (2001); morphology Reduced concentrations of DNA in jejunum, Leenhouwers et al. (2006) ileum, and liver, indicating programmed cell death Augmented concentrations of RNA in the colon Reduced villi length Increased depth of intestinal crypts in jejunum and ileum Impaired water absorption, can lead to dehydration Increased rate of turnover of intestinal mucosal cells Alteration in the native gut Stimulated microbial fermentation in Wood and Serfaty-Lacrosniere (1992); Choct (1997); Amirkolaie et al. (2006); microflora intestine. Leenhouwers et al. (2007a, 2007b) Enhanced volatile fatty acids, such as acetic acid, propionic and butyric acids, production Lower pH of intestinal tract; in long term may disturb the normal micrbiota prevailing in gut Influenced bioavailability of dietary minerals Decreased oxygen tension, favouring development of anaerobic microbiota Alteration in gut mucus Increased concentrations of lumenal mucin in Satchithanandam, Klurfeld, Calvert, and Cassidy (1996) layer stomach and small intestine 1416 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 removal of essentially all starch. Moreover, during the same decade holding capacity and assist in faecal bulking in non-ruminant Theander and Åman (1982) developed an indirect method to animals (Pluske, Kim, McDonald, Pethick, & Hampson, 2001). The analyse total dietary fibre (TDF) in food stuff by quantifying the factors associated with the detrimental effects of NSPs are discussed amounts of uronic acids, sugars, Klason lignin and starch and then below. calculating the TDF as the sum of the uronic acids, sugars and Klason lignin, minus the concentration of starch. In contrast, 6.1. Modulation in digesta viscosity Prosky et al. (1984) developed a direct method of TDF quantification. These assays have been further expanded to allow The solubility and molecular weight of NSPs determine the vis- quantification of both soluble and insoluble dietary fibre compo- cosity. The solubility is not specific to the sugar composition or nents (Theander, Åman, Westerlund, Anderson, & Pettersson, linkage present in NSPs but depends on the chemical structure 1995) and refined to increase precision and decrease the complex- and association of NSPs with the cell wall components. However, ity and time required (Lee, Vincet, Prosky, & Sullivan, 1996). the physical effect of viscosity on digestion and absorption of nutrients appear to be similar regardless of the sources of NSPs. More- over, the binding of NSPs with the intestinal brush border increases 5.2. Monomeric component analysis the thickness of the unstirred water layer adjacent to the mucosa, leading to impaired nutrient digestion and absorption (De Lange, Monomeric component analysis was first developed by Englyst 2000). Furthermore, increased endogenous intestinal secretion of and Cummings (1988). In this method all starch is hydrolysed water, nutrients and other electrolytes has been suggested as a enzymatically and NSPs are measured as the sum of the constitu- cause of reduced nutrient digestion (Choct, 1997). High viscosity ent sugars released by acid hydrolysis. The individual sugars are also increases residence time of the digesta and therefore increases subsequently quantified by gas chromatography (GC) or by intestinal volatile fatty acid (VFA) production. The resulting drastic high-performance liquid chromatography (HPLC) (Englyst, King- changes in the gut ecosystem decrease nutrient digestion and man, & Cummings, 1992; Englyst, Quigley, & Hudson, 1994). eventually reduce performance (Choct et al., 1996). In rainbow Moreover, a single value for total sugars may be obtained by a col- trout (Oncorhynchus mykiss), reduced nutrient digestibility on orimetric procedure that measures NSPs as reducing sugars (Eng- inclusion of dietary soluble NSP (guar gum) was associated with lyst et al., 1994). The GC technique for dietary fibre analysis, an increase in digesta viscosity (Storebakken, 1985). Similarly, preferred by many researchers, measures NSP as the sum of neu- inclusion of soybean NSPs in the diet of Atlantic salmon resulted tral sugars obtained by GC and uronic acids measured separately in a relatively high viscosity in the intestinal content that was re- (Mongeau et al., 2001). In this procedure, the sugars are reduced flected in the reduction of amino acid and lipid digestion (Refstie to their alditols with alkaline sodium borohydride and acetylated et al., 1999). Cumulative apparent absorption of amino acids, nitro- with acetic anhydride in the presence of methylimidazole as cat- gen, and sulphur was slowed down in Atlantic cod (Gadus morhua) alyst. Since the uronic acid-containing polysaccharides are more by dietary bioprocessed soybean meal, which was possibly due to difficult to hydrolyse and require treatment with concentrated the high water-binding capacity of this soy product (Refstie et al., acid at high temperature; they are measured separately by color- 2006). imetry (Scott, 1979). The HPLC method of dietary fibre analysis Feeding tilapia (Oreochromis niloticus) with cereal grain NSPs (Englyst et al., 1994) is very common and measures NSPs as the led to augmentation in digesta viscosity and reduction in digesta sum of neutral sugars and uronic acids, directly by electrochemi- dry matter. Moreover, the absorption of minerals, especially so- cal detection. dium ion, was significantly negatively correlated with digesta It must be noted that the values obtained by GC or HPLC are viscosity (Almirall, Francesch, Pe´rez Vendrell, Brufau, & Esteve- typically lower in comparison to the gravimetric method of analy- Garcı´ a, 1995; Leenhouwers, Ortega, Verreth, & Schrama, 2007b). sis because of the exclusion of lignin and resistant starch during Similarly, inclusion of soluble NSPs in the diet of African catfish chromatographic assay. The application of near infra-red reflec- (Clarias gariepinus) induced large increases in digesta viscosity tance (NIR) or transmission (NIT) spectroscopy for rapid estimates and thereby intestinal fermentation activity, digesta dry matter of non-starch polysaccharides are in vogue (Blakeney & Flinn, content and digestive organ weights and the nutrient digestibil- 2005). Near infra-red reflectance spectroscopy provides fast, safe, ity was adversely affected beyond a certain viscosity threshold and inexpensive analysis. It is, however, a comparative technique (Leenhouwers, Adjei, Verreth, & Schrama, 2006; Leenhouwers, that relies on multivariate calibration of sample spectra and accu- Ter, Verreth, & Schrama, 2007a). In addition, supplementation rate reference analysis (Neas, Isaksson, Fearn, & Davies, 2002; of galactomannan-rich endosperm of sesbania (Sesbania aculeate) Williams & Norris, 2001). It has the potential to be exploited as a seeds in the diet of common carp (Cyprinus carpio) resulted in in- rapid analytical method for nutritionally important components, creased viscosity in the intestinal content, thereby affecting the including polysaccharides. nutrient absorption and utilisation (Hossain, Focken, & Becker, 2001). In general, the soluble NSPs are generally viscous in nat- 6. The anti-nutritive effect of non-starch polysaccharides ure, and therefore they enhance the viscosity of the diet as well as intestinal digesta. The magnitude of viscosity development in The enzymes for NSP digestion such as b-glucanases and b-xylan- gut with response to dietary NSPs varies among animals and also ases are scarce or not present in fish (Kuz’mina, 1996). Consequently, depends on the source of NSP (Montagne, Pluske, & Hampson, the dietary NSPs remain undigested and therefore negatively affect 2003). Digesta viscosity in the proximal intestine of African cat- animal performance. The adverse effect is associated with the vis- fish and Nile tilapia was considerably higher in groups fed rye cous nature of NSPs, their physiological and morphological effects than in those fed maize and wheat (Leenhouwers et al., 2007a, on digestive tract, interaction with epithelium, mucus and micro- 2007b). flora of gut (Table 3). Subsequently, it has been reported that soluble The age of fish may be an important factor determining NSPs, such as mixed-linked b-glucans present predominantly in oats their sensitivity towards viscosity. The probable reason for this and barley, increase intestinal transit time, delay gastric emptying age-related reaction is that a more developed intestinal micro- and glucose absorption, increase pancreatic secretion, and slow biota in older animals is able to utilise NSPs more efficiently absorption. However, the insoluble NSPs, like pentosans (arabinoxy- (Choct & Kocher, 2000; Refstie et al., 2006a) than in younger lans and xylans), beneficially decrease transit time, enhance water- ones. A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1417

6.2. Alteration in gastric emptying and rate of passage cantly greater (Jin, Reynolds, Redmer, Caton, & Screnshaw, 1994). These authors also reported enlargement in the width of intestinal Dietary soluble NSPs increase the viscosity of digesta in mono- villi and increased depth of intestinal crypts in jejunum and ileum, gastric animals and decrease the rate of passage, whereas the and increased rate of cell proliferation and increased crypt depth insoluble NSPs, such as cellulose and hemicellulose, increase the in the large intestine. Since the crypts are the principal sites of cell passage rate (Johansen, Knudsen, Sandstrom, & Skjoth, 1996). proliferation in the intestinal mucosa (Baserga, 1985), an increase Dietary inclusions of soluble NSPs reduce the rate of gastric in the rate of crypt-cell proliferation, along with the reduction in emptying in fish, which can delay the intestinal absorption of concentrations of DNA, may increase the rate of turnover of intesti- glucose (Bach Knudsen, 2001) and possibly of other nutrients. In nal mucosal cells. It could be an explanation for the reduction in vil- African catfish, inclusion of viscous cereal grains in the diet re- lus height and crypt depth ratio observed in the small intestine of duced the plasma cholesterol and glucose levels (Leenhouwers pigs subjected to feed supplemented with high fibre (Jin et al., et al., 2007a). In common carp fed diets containing sesbania endo- 1994). Similarly, administration of an oral dose of cellulose to pre- sperm, both muscle and plasma cholesterol levels were signifi- weaning pigs reduced villus length by approximately 15% in the jeju- cantly lowered (Hossain et al., 2001). Also significant reduction num and ileum (Jin, 1992). Furthermore, McDonald (2001) reported in total cholesterol levels in blood plasma of rainbow trout, yellow- that addition of sodium carboxymethylcellulose (CMC) to a highly tail and Atlantic salmon fed diets containing NSP-rich soybean digestible cooked rice-based weaner diet (40 g/kg diet dry matter) meals has been reported by various authors (Kaushik et al., 1995; for 13 days significantly increased the intestinal viscosity of digesta Refstie et al., 1999; Shimeno, Hosokawa, YÅmane, Masumoto, & within the small and large intestine, which led to decreased villus Uneno, 1992). A reduced blood cholesterol level is probably associ- length and increased crypt depth. Similar results have been reported ated with binding or trapping of bile salts in the gut, due to high by Hopwood, Pethick, and Hampson (2002). viscosity, as also observed in rats fed with galactomannans from The shortening of villi results in an impaired absorption because guar gum (Moundras, Behr, Remesy, & Demigne, 1997). shortening results in an absolute loss of intestinal surface area and cells. Since the osmotic water absorption is governed by nutrient absorption, the decline in nutrient absorption caused by reduction 6.3. Alteration of gut physiology, gut morphology, native gut of villi length eventually impairs the water absorption. Moreover, microflora and mucus layer of gut an increased crypt depth is associated with an increased water secretion into the intestinal lumen. This large fluid amount in the Apart from increasing the gut viscosity, the soluble NSPs elicit intestinal lumen is generally not absorbed by the partially devel- an anti-nutritive effect by modifying the gut functions. These ham- oped large intestine of young animals, which may give rise to clin- per the endogenous secretion of water, proteins, electrolytes and ical symptoms of dehydration (Nabuurs, 1998). In chicken, feeding lipids (Angkanaporn, Choct, Bryden, Annison, & Annison, 1994). with gum xanthan-supplemented diet for 14 days resulted in Non-starch polysaccharides can enhance bile acid secretion and deepening of the jejunal crypts and reduction in villi height and subsequently result in significant loss of these acids in the faeces villi surface. Additionally, the viscous NSPs increased the weight (Ikegami et al., 1990). This can result in increased hepatic synthesis of the small intestine. However, changes in the weight of visceral of bile acids from cholesterol to re-establish the homeostasis, organs were generally due to variation in rate of cell proliferation, which may ultimately influence the absorption of lipids and cho- cell size or protein synthesis (Iji et al., 2001). This was further con- lesterol in the intestine, resulting in lower blood cholesterol levels firmed in this study by an increase in the protein:DNA ratio (cell (Hossain et al., 2001). These impacts could lead to remarkable size) of the jejunum and by a decline in the DNA content (cell pop- changes in the dynamics of the gut physiology, ensuring poor ulation) of chickens raised on NSP-containing diets. nutrient assimilation efficiency by the animal. Non-starch polysac- Conversely, Moore, Kornegay, Grayson, and Lindemann (1988) charides may also influence lipid metabolism in the intestine, found that villus shape or surface morphology in jejunum of grow- through binding with bile salts, lipids and cholesterol (Vahouny, ing pigs was not affected by a high-fibre diet, although some loss of Tombes, Cassidy, Krichevsky, & Gallo, 1981). Hitherto, there are epithelial cells at the apex of the villi was observed. In rats, inges- no reports on the direct inhibition of intestinal enzyme synthesis tion of pectin (25 g pectin/kg of elemental diet for 14 days) signif- by NSPs but the activities of most enzymes may be reduced icantly increased villus height and crypt depth (Andoh, Bamba, & through coupling to NSPs or physical restriction of enzyme access Sasaki, 1999). Supplementation of low viscosity CMC (40 g CMC/ to substrates (Pettersson & Åman, 1989). Digestive enzyme activity kg air-dried diet) to a cooked rice-based diet increased the small of broiler chicks responded to diets supplemented with commer- intestinal villus and crypt depth in newly weaned pigs without cial NSPs (Iji, Saki, & Tivey, 2001). The jejunal maltase and sucrase altering the shape of villi (McDonald, 2001).The above studies activities were highest in chicks that were fed an alginic acid-sup- highlight the effects of NSPs on gut morphology of monogastric plemented diet (low viscosity) and lowest in chicks fed the gum animals. It is also evident that information on fish is lacking. xanthan-supplemented diet (high viscosity). The activity of amino- Mechanisms for modifying gut morphology: The consumption of peptidase N in the ileum was also stimulated by a highly viscous an NSP-rich diet alters the intestinal physiology and anatomy by NSP diet. However, the uptake of amino acid L-tryptophan into its ability to increase digesta viscosities. The rates of crypt-cell pro- brush-border membrane vesicles was unaffected by NSP supple- liferation, cell migration along the crypt–villus axis, and cell extru- ment. To our knowledge, no information is available on the direct sion from the villous apex via apoptosis and cell sloughing control effects of NSPs on fish gut physiology. the dynamic process of small intestinal cell turnover. The presence A number of authors have reported that dietary NSPs have a con- of high digesta viscosity in the lumen may increase the rate of vil- siderable impact on the gut anatomy and gut development. A pro- lus cell losses, leading to villus atrophy, a phenomenon associated longed consumption of soluble NSPs is associated with increased with an increased crypt-cell production, and generally with in- size and length of the digestive organs in pigs (McDonald, 2001), creased crypt depth (Montagne et al., 2003). chickens (Iji et al., 2001) and fish (Leenhouwers et al., 2006) accom- The delay of digesta passage in the intestinal tract, as a result of panied by a decrease in nutrient digestion. Pigs fed with high dietary increase in viscosity, may stimulate microbial fermentation in the fibre food for 14 days resulted in significant reduction in concentra- intestine. The fermentation of NSP produces volatile fatty acids tions of DNA in jejunum, ileum, and liver, indicating programmed (VFA) as an end product. Administration of NSPs in the diet of tila- cell death, while concentrations of RNA in the colon were signifi- pia and African catfish has been shown to increase VFA level in the 1418 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 intestinal tract (Amirkolaie, Verreth, & Schrama, 2006; Leenhouw- erals. This is because of a reduction in the rate of gastric emptying, ers et al., 2007a, 2007b). Acetic acid is the prominent VFA produced leading to a depression in nutrient absorption (Bach Knudsen, in African catfish, which is in agreement with other studies on tila- 2001). Moreover, Leenhouwers et al. (2006) explained that reduced pia and marine herbivores (Amirkolaie et al., 2006; Clements & digestibility of nutrients is related to partial distribution of diges- Choat, 1995; Kihara & Sakata, 1997). Besides acetic acid, propionic tive enzymes in a viscous solution and a lowered flow at the muco- and butyric acids are also produced following microbial fermenta- sal layer. Enhanced endogenous losses of nutrients and increase in tion in fish but the concentration of these two acids varies among the thickness of the unstirred water layer adjacent to the mucosa fish species. A low concentration of propionic and butyric acid in also lead to the diminution in nutrient digestion and absorption tilapia was observed (Amirkolaie et al., 2006) while a significant (De Lange, 2000). high amount of these acids was reported from marine herbivorous fish (Clements & Choat, 1995). The production of these organic 7.1. Effect on glucose acids in the intestinal tract may lower its pH, which in the long term may disturb the normal microbiota prevailing in the gut. Be- The inclusion of NSPs in the basal diet of monogastric animals sides, gastric acidity influences the bioavailability of dietary miner- including fish has been reported to delay the intestinal absorption als (Wood & Serfaty-Lacrosniere, 1992) by regulating chelation and of glucose. In African catfish it was demonstrated that feeding diets complex formation and by altering the transport mechanisms of containing rye at a level of 400 g/kg diet (dry matter) decreased minerals (Ravindran & Kornegay, 1993). Moreover, increase in plasma glucose level (Leenhouwers et al., 2007a). In salmonid fish, the residence time of digesta in the intestine following intake of inclusion of guar galactomannans and alginates as NSP sources in soluble NSPs may decrease oxygen tension and favour the develop- the diet reduced the availability of glucose when compared to ment of anaerobic microbiota (Choct, 1997). Although it is not fully NSP-free diets (Storebakken, 1985; Storebakken & Austreng, known whether a sudden change of gut ecology is detrimental to 1987). Significantly lower intestinal maltase activity in Atlantic the efficiency of nutrient utilisation, the maintenance of health sta- salmon has been reported on feeding defatted soybean meal con- tus relies mainly on normal endogenous microbiota. The normal taining NSPs at a level of 100 g/kg diet (dry matter) (Kraugerud microbiota confers many benefits to the intestinal physiology of et al., 2007). In fish, knowledge on the glucose digestibility in the host. Some of these benefits include the metabolism of nutri- response to dietary NSPs is limited and only few studies have been ents and organic substrate and the contribution to the phenome- conducted to elucidate the effect of NSP on carbohydrate metabo- non of colonisation resistance. However, when the delicate lism and absoption. However, a considerable number of reports are balance of normal microbiota is upset, pathogens that arrive or available in pigs supporting a negative effect of dietary NSP on glu- that have already been present but in numbers too small to cause cose level. In growing swine the use of guar gum in basal feed disease take the opportunity to multiply. Furthermore, the prolifer- halved the rate of absorption of glucose in the jejunum (Rainbird, ation of some anaerobic organisms can lead to production of toxins Low, & Zebrowskat, 1984). A reduction of 25% in the plasma glu- and deconjugation of bile salts which are essential for the digestion cose concentration in pigs fed with semipurified diets supple- of fat (Carre, Gomez, & Chagneau, 1995). mented with 40 g/kg guar gum in the diet (dry matter) has also Mucus is the protective layer of the entire gastro-intestinal been reported (Sambrook & Rainbird, 1985). Furthermore, Nunes tract, which is exposed to all chemical and physical forces of diges- and Malmlof (1992) demonstrated that feeding 60 g/kg guar gum tion. Several studies have correlated the dietary NSPs intake and in the diet to swine reduced glucose absorption by 32%. Further- mucin concentration in the gastro-intestinal tract. In pigs, inclu- more, the inclusion of 60 g/kg guar gum in semipurified diet of sion of pea fibre in a wheat diet tended to increase the output of swine reduced the postprandial production of insulin by 30%, insu- mucins in the ileal digesta from 6.1 to 7.3 g per day for diets sup- lin-like growth factor-1 (IGF-1) by 58%, gastric inhibitory polypep- plemented with 0 and 240 g of pea fibre per day, respectively (Lien, tide by 55% and glucagon by 41% (Nunes & Malmlof, 1992). Lower Sauer, & He, 2001). Similarly ileal glucosamine and galactosamine blood glucose level was observed when trout were fed an experi- excretion increased continuously with fibre intake (Reverter, mental diet prepared by replacing 62.5% of fish meal protein with Lundh, & Lindberg, 1999). When wheat bran (150 g/kg diet dry detoxified Jatropha curcas kernel meal (DJKM) which contained matter) was added to a protein-free diet for pigs, the ileal output 16% NSP (Kumar, Makkar, & Becker, 2010). of galactosamine at the terminal ileum increased from 1.93 to 4.13 g per day (Fuller & Cadenhead, 1991). Feeding CMC as a diges- 7.2. Effect on protein ta viscosity-inducing non-fermentable polysaccharide to weaned piglets for 15 days significantly increased viscosity of ileal digesta Inclusion of NSPs in the diet of fish has been well documented to and ileal mucin concentration (Piel, Montagne, Sève, & Jean-Paul, reduce amino acid digestibility. The decrease in nitrogen utilisation 2004). The mechanisms by which dietary NSPs modify mucin char- efficiency following an NSP-rich diet is probably due to increase of acteristics are not well understood. The physical scraping and pro- N secretion either endogenously and/or through intestinal bacteria. teolytic breakdown of mucus gels are the main factors releasing Leenhouwers et al. (2006) reported that inclusion of soluble NSPs mucins into the gut lumen (Allen, 1981). Therefore, it could be from guar gum at a level of 40 and 80 g/kg in the diet (dry matter) hypothesised that the erosion of mucus layer may be due to an in- of African catfish diet significantly increased digesta viscosity in the crease in the bulk of digesta, stretching the intestinal mucosa and proximal and distal intestine. This increase in viscosity was accompa- scraping mucin from the mucosa as they pass through the digestive nied by a reduction in the apparent digestibility coefficient of protein. tract. Storebakken (1985) reported reduced protein digestibilities in rain- It could be contemplated that effects reported above may also bow trout fed with 25–100 g guar gum per kg diet. African catfish appear in fish but to date no such studies have been conducted reared with a high-viscosity rye-supplemented diet lowered the pro- in fish. tein digestibility much more than those fed with a low-viscosity wheat-based diet (Leenhouwers et al., 2007a). However, it was in contrast with the findings of Leenhouwers et al. (2007b) with tilapia 7. Effect of non-starch polysaccharides on nutrient metabolism where reduction in protein digestibility was observed more in fish fed with low-viscosity wheat than high-viscosity rye-based diet. This Feeding of NSPs negatively influences the metabolism and util- suggests that viscosity is not the only factor that explains the differ- isation of dietary nutrients, like glucose, lipid, amino acid and min- ences in protein digestibility in Nile tilapia. This species of fish is less A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1419 sensitive to viscous dietary ingredients than African catfish, due to its Cu, Fe and K utilisation were observed (Kraugerud et al., 2007). more herbivorous feeding habit (Leenhouwers et al., 2007b). Simi- Furthermore, in rainbow trout Na excretion has been found to larly, feeding salmon on soya diets with high levels of soluble NSPs re- increase proportionally with dietary cellulose level (Øvrum & sulted in reduced protein digestibilities (Refstie et al., 1999). Storebakken, 2007). In contrast to the above findings, no correla- Furthermore, sesbania endosperm, a leguminous seed at P7.2% in tion was observed between NSP content in diet and sodium diet of common carp (Hossain et al., 2001)andatP5.8% in diet of tila- excretion in Atlantic salmon (Aslaksen et al., 2007). pia (Hossain, Focken, & Becker, 2003) substantially reduced the pro- Although dietary NSPs have mainly been reported to have neg- tein efficiency ratio and protein productive value. Other studies that ative effects on mineral utilisation, epidemiological data suggests have included purified soluble NSPs in fish diets also found reduced that intake of plant ingredients rich in NSPs can be a protective fac- protein digestibilities (Shiau, Yu, Hwa, Chen, & Hsu, 1988). tor against the abnormalities caused by metal toxicity. Despite a number of studies conducted to elucidate the effects of dietary 7.3. Effect on lipid and cholesterol level NSPs on mineral bioavailability, little attention has been devoted to the understanding of their chelating mechanism. Increasing the NSP content in the diet of monogastric animals has been reported to decrease the utilisation of lipid. Increase in digesta viscosity caused by intake of an NSP-containing diet has 8. Effect on growth performance and body composition been shown to affect emulsification negatively, and to reduce lipolysis (Pasquier et al., 1996). Non-starch polysaccharides may entrap Low-protein soy products have been shown to induce negative bile salts, thus reducing their efficiency in solubilising fats and con- effects on the digestibility of nutrients in salmon, which was prob- sequently impairing lipid absorption (Ebiharam & Schneeman, ably an effect of the viscosity caused by the NSP in the soybean 1989). In African catfish, the digestibility of fatty acids was reduced product (Refstie et al., 1999). Besides, in rainbow trout, tilapia considerably, when fed with wheat or rye at a level of 400 g/kg of and Atlantic salmon, an increased water content of the gut induced basal feed (dry matter) (Leenhouwers et al., 2007a). Feeding of by the water-binding properties of dietary soluble NSP has been common carp with sesbania endosperm (containing about 75% of suggested as the reason for the reduced nutrient digestibilities galactomannan) at levels of P7.2% significantly reduced both mus- and diminished growth performance (Refstie et al., 1999; Shiau cle and plasma cholesterol levels (Hossain et al., 2001). Similar ef- et al., 1988; Storebakken, 1985). Feeding rainbow trout a diet con- fects were observed when tilapia was fed sesbania endosperm at taining 10% guar gum, which contains high proportions of galacto- levels of P5.8% (Hossain et al., 2003). Moreover, reduced total cho- mannan, resulted in significant reduction of growth and dry matter lesterol levels in blood plasma of Atlantic salmon fed diets contain- and fat content in fish tissues. Moreover, in common carp and trout ing soybean meal have also been reported (Refstie et al., 1999), the replacement of 75% and 62.5% of FM protein by DJKM respec- which could possibly be ascribed to NSPs present in soybean meal. tively decreased growth performance. This adverse effect was Similarly, 62% of FM protein replacement by DJKM (detoxified attributed to the presence of NSPs in DJKM (Kumar, Makkar, & jatropha kernel meal) resulted in lower lipid digestibility in trout, Becker, 2008; Kumar et al., 2010). Dietary inclusion of sesbania which could possibly be due to the presence of NSPs in DJKM endosperm at levels of 7.2%, 10.8% and 14.4% in the feed of com- (Kumar et al., 2010). The presence of NSPs in feeds has been mon carp resulted in reduced body weight gains that were 57%, hypothesised to reduce fat absorption in trout by disturbing mi- 48% and 39%, respectively of the control diet (Hossain et al., celle formation in the gastro-intestinal tract (Øverland et al., 2009). 2001). Additionally, the inclusion of sesbania endosperm influ- The hypocholesterolaemic response on NSP intake has also been enced whole body proximate composition of fish, with signifi- reported in Atlantic salmon (Salmo solar) fed with soybean meal cantly increased whole body moisture, reduced lipid and gross containing NSPs at a level of 100 g/kg diet (dry matter) (Kraugerud energy content (Hossain et al., 2001). Moreover, similar observa- et al., 2007). This is probably associated with binding of cholesterol tions of higher whole body moisture and lower lipid content in with bile salts in the gut. Moreover Potter (1995) described an- common carp fed diets containing various levels of rape seed, mus- other possible mechanism for hypocholesterolaemia, as an tard oil-cake, linseed and sesame meal have been made (Dabrow- enhancement of bile acid excretion and consequently creation of ski & Kozlowska, 1981; Hossain & Jauncey, 1989). The study of an environment in which cholesterol is being ‘pulled’ from the Hossain et al. (2001) showed no significant difference in the body. In this state, hepatic cholesterol metabolism alters to provide hepatosomatic index of fish. It is in agreement with other studies cholesterol for enhanced bile acid synthesis. on fish and poultry in which the effects of feeding soluble NSPs on relative liver weight were insignificant (Iji et al., 2001; Leen- 7.4. Effect on minerals houwers et al., 2006; Storebakken, 1985). Conversely, the study conducted on African catfish showed stimulating effects of guar Various components of NSPs interact with minerals and have gum on stomach and intestine weights (Leenhouwers et al., been shown to decrease mineral absorption. Components of poly- 2006). A feeding trial on tilapia showed that weight gains of fish saccharides and lignin that interact with minerals include the car- fed diets containing 5.8%, 8.7% and 11.8% sesbania endosperm boxyl group of uronic acid, carboxyl and hydroxyl groups of were 82%, 73% and 64%, respectively compared to the control diet phenolic compounds and the surface hydroxyl of cellulose (Torre, (Hossain et al., 2003) and these fish also had significantly higher Rodriguez, & Sauracalixto, 1995). Moreover, NSP-induced digesta whole body moisture, lower lipid and lower gross energy contents. viscosity has been shown to hinder mineral absorption (Van der It was also noticed that the reduction in whole-body lipid was Klis, Kwakernaak, & De Wit, 1995). Absorption of Ca, Mg, Na and more pronounced in the diets containing higher levels of endo- P were considerably lower when African catfish were fed with a sperm. It may be because of the thickening of the fluid layer closest diet supplemented with rye (Leenhouwers et al., 2007a), while to the mucosal wall, thereby preventing contact of digestive only Na absorption was significantly reduced when the same diet enzymes with the substrates and the formation of the micelles re- was fed to Nile tilapia (Oreochromis niloticus)(Leenhouwers et al., quired for the lipid absorption (Wang, Newman, Newman, & Hofer, 2007b). Elevated faecal sodium excretion in Atlantic salmon as 1992). In contrast to the above findings, Leenhouwers et al. (2007a) an effect of feeding soy products was pointed out by Storebakken reported no effect on the growth performance in African catfish et al. (1998). Likewise, in the same fish species a negative impact with increase in intestinal viscosity. A previous study with African of defatted soyabean meal on K, Na, Zn and of native soy-NSP on catfish also found that the guar-gum-induced changes in digesta 1420 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 characteristics were not accompanied by reduced fish performance gests that the anti-nutritive activity of NSPs in poultry is related to (Leenhouwers et al., 2006). These differences could be attributed to the gut microflora, as addition of antibiotics to diets increased factors such as differences in NSP concentration, NSP structure, nutritive value of diets (Langhout & Schutte, 1995). Moreover, in concentration of other dietary components, fish species, and age swine, supplementation of virginiamycin at a level of 11 ppm in of fish (Leenhouwers et al., 2007a; Petersen, Wiseman, & Bedford, diets containing 50% oats improved dry matter, energy, hemicellu- 1999). lose and cellulose digestibilities. Furthermore, the antibiotic supplementation also improved absorption and retention of minerals, such as Ca, P, Mg, Cu, Fe, Zn and Mn (Ravindran & Korne- 9. Non-starch polysaccharides and effect on gelatinisation gay, 1993). Similarly, 0.7% nebacitin in a pig diet containing 10.5% fibre enhanced the apparent N digestibility by 12%; salinomycin at Fish in general have a limited capacity for carbohydrate utilisa- a level of 82 mg/kg of a diet (dry matter) containing 20% wheat tion and processing methods, such as gelatinisation, have been bran increased P absorption (Moore, Kornegay, & Lindemann, reported to improve the nutrient bioavailability to the fish. Gela- 1986). Only a very limited number of works has been done on tinisation is a thermal modification of raw dietary carbohydrates. the interactions of NSPs and antibiotics in fish. In Atlantic salmon, During this process carbohydrate granules are modified in such a soybean meal-induced enteritis was not ameliorated by adding the way that their susceptibility to enzymatic action increases (Kumar, broad-spectrum antibiotic oxytetracycline to the diet at 3 g/kg (dry Sahu, Pal, Choudhury, & Mukherjee, 2006), making digestion more matter) (Bakke-McKellep et al., 2007). In the distal intestinal mu- complete. Carbohydrate gelatinisation and its impact on digestibil- cosa of fish fed both soybean meal and soybean meal supple- ity and growth have been well documented in fish such as carp mented with oxytetracycline, a significant increase in the (Kumar et al., 2006) and rainbow trout (Podoskina, Podoskin, & proliferative compartment length as well as apparent increases in Bekina, 1997). Wheat, being the major source of starch, also con- the number of cells undergoing cellular repair processes and apop- tains a low amount of NSPs. Arabinoxylan, the main NSP in wheat tosis were observed as pathophysiological responses related to flour, is reported to hinder the gelatinisation and retrogradation enteritis induced by the soybean meal (Bakke-McKellep et al., properties of wheat starch, due to its water-binding capacity and 2007). In the same species, it was seen that soybean meal supple- high viscosity (Shogren , Hashimoto, & Pomeranz, 1987). Moreover, mented with oxytetracycline resulted in reduced relative liver addition of an isolated NSP-rich fraction to starch markedly affects weight and did not modify the responses to dietary soybean meal starch gelatinisation and pasting properties (Sasaki, Yasui, & influencing gut morphology, hydrolysis of protein and starch, and Matsuki, 2000). Furthermore, Tester and Sommerville (2003) absorption of amino acids, nitrogen and sulphur (Refstie et al., reported that soluble NSPs restrict the gelatinisation of maize 2006b). starch and wheat starch by reducing hydration of the amorphous regions and consequently limiting depression of the glass transi- tion temperature within these regions. This suggests that the effect 11. Purified non-starch polysaccharides as immunostimulants of NSPs on the gelatinisation event, in general is mediated via reducing the volume fraction of water in the system and restricting Immunostimulants are chemical substances that activate the water availability and mobility due to the hydration process generalised immune response system of animals. Such substances (Tester & Sommerville, 2003). The plant tissues containing starch are also known to render animals more resistant to infectious dis- are expected to contain NSPs, which could hinder the effect of eases and reduce the risk of disease outbreaks if administered prior processing on starch digestibility. Better understanding of the to stress-causing situations. NSP, such as b-1,3-glucans, acts as an effects of different NSP types on gelatinisation would aid in the immunostimulant and thus improves health, growth and general efficient utilisation of plant carbohydrates in fish nutrition. performance of many different animal groups, including farmed shrimp, fish and terrestrial animals (Kumar et al., 2005). Supple- 10. Interaction with antibiotics mentation of b-1,3 glucan in diets enhances non-specific cellular defence mechanisms by increasing the number of phagocytes The efficiency of NSP utilisation by fish is dependent on the nat- and the bacterial killing activity of macrophages in rainbow trout, ure of the microbial population residing in the gut. It, could, there- Atlantic salmon, catfish, and carp, and also by the macrophage fore, be speculated that the supplementation of antibiotics that mediated superoxide anions production (Kumar et al., 2005). In a alter the gut microflora would have an influence on dietary fibre major Indian carp, Labeo rohita, yeast glucan (b-1-3 glucan) has utilisation (Annison & Choct, 1991). A large body of evidence sug- been observed to enhance the phagocytic activity of leucocytes

Table 4 Role of b-glucan in health and production of ﬁsh and crustaceans (adapted from Kumar et al., 2005).

Fish/crustacean Route of administration Advantage

Fish Feed or injection Enhances production of antibodies against pathogens Acts in synergy with antibioticsEnhances resistance to bacterial disease and efficacy of vaccine Fish and shrimp Injection and oral Acts as a true adjuvant and enhances antibody production Halibut larvae Immersion Increases survival rate Coho salmon Injection or oral Protects against Aeromonas salmonicida Shrimp Feed Increases growth, reduces mortality, and increases feed utilisation Fish and shell fish Feed Acts in synergy with vitamin-C Shrimp post larvae Immersion Enhances the survival rate Penaeus monodon Feed Protects against white spot syndrome virus infection Increases survival rate Macrobrachium rosenbergii larvae Bath treatment (10 mg/L) Increases lysosomal activity Protects against Vibrio alginolyticus Carp Injection (500 mg/kg) Enhances protection against Aeromonas hydrophila Asian catfish Feed Enhances immunity against Aeromonas hydrophila A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1421

Table 5 Non-starch polysaccharide degrading enzymes in animal feed and their impact (adapted from Bhat, 2000).

Enzyme Effect Impact

Cellulases and hemicellulases Partial hydrolysis of lignocellulosic materials Improvement in nutritional quality of animal feed Hydrolysis of b-glucans Improvement in performance of ruminants and monogastrics Decrease in intestinal viscosity Better emulsiﬁcation and ﬂexibility of feed materials b-Glucanase and xylanase Hydrolysis of cereal b-glucans and arabinoxylans Improvement in feed digestion and absorption, and in weight gain of Decrease in intestinal viscosity broiler chickens and hens Release of nutrients from grains Hemicellulase with high Increase in nutritive quality of pig feeds Reduction in the cost of pig feeds xylanase activity Reduction in the cost of feeds for pigs Cellulases, hemicellulases and Partial hydrolysis of plant cell wall during silage and Contribution to production and preservation of high quality fodder for pectinases fodder preservation ruminants Improvement in quality of grass silage

and stimulates generation of reactive oxygen species (ROS) in also reduce the effect on those bacteria that are resistant to phagocytes (Ali, Karunasagar, Pais, & Tauro, 1996). A number of phagocytosis. studies have shown that b-1-3/1-6-glucans enhance the biological Mannose units that comprise glucomannans, belong to the cat- activity of shrimp haemocytes and improve growth, survival rate egory of compounds that adhere to receptors used by pathogenic and feed conversion efficiency under experimental conditions microbes as the first step of colonisation of the gut. Therefore, sup- (Table 4). b-glucans have been successfully used to increase the plementation of mannose in basal feed of animals may contribute resistance of shrimp Penaeus japonicus against vibriosis (Kumar to better health by interfering with colonisation and growth of et al., 2005). Penaeus monodon when fed with b-glucan at 0.2% pathogens in the gut (Raa, 2000). (w/w of the feed) significantly increased phenol oxidase, the num- Levan, a natural polymer of fructose with b (2,6) linkages pro- ber of haemocytes and the bacterial killing activity against vibrio- duced extracellularly by many microorganisms like Zymomonas sis, white spot syndrome virus, Vibrio damsela and V. harveyi and mobilis, Bacillus subtilis, Bacillus polymyxa, and Acetobacter xylinum, also enhanced survival and immunity during brood stock rearing is shown to have immunostimulating properties in fish (Gupta (Chang, Su, Chen, & Liao, 2003). Macrobrachium rosenbergii post lar- et al., 2008; Rairakhwada et al., 2007). Dietary levan supplements val stage showed enhanced growth and resistance to V. alginolyti- at a concentration of 0.5% have been reported to activate non-spe- cus as a result of dietary administration of b-glucan (Misra et al., cific phagocytes in Cyprinus carpio juveniles which resulted in 2004). However, the long term administration of b-glucan in fish higher survival rate when challenged with Aeromonas hydrophila and shellfish can reduce the immune response to basal levels and (Rairakhwada et al., 2007). Likewise the histological study strongly

Fig. 6. Modes of action of non-starch polysaccharide degrading enzymes (Wyatt, Parr, & Bedford, 2008). 1422 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 suggests that levan supplementation at 1.25% ameliorates the ef- ports three types of mechanisms could be suggested for the action fects of infection by A. hydrophila in Labeo rohita juveniles. of NSP-degrading enzymes (Fig. 6). These are discussed below.

12. Non-starch polysaccharides degrading enzymes 12.1.1. Disruption of cell wall integrity The cell wall in cereals and legumes is constructed mainly of Monogastric animals including fish lack the intestinal enzymes small amounts of cellulose, hemicellulose and arabinoxylan with for the degradation of NSPs. The improvement in the digestibility minor b-glucan components. The activity of exogenous NSP- of NSPs is achieved by supplementation of NSP-degrading enzymes degrading enzymes creates ‘holes’ in the cell wall. This allows in the diet. Such an approach has successfully been used in poultry water hydration and permits pancreatic proteases and amylases diets but little work showing the potential of NSP-degrading en- to act, enabling better digestion of the starch and protein. Xylan- zymes in fish is available (Ai et al., 2007). ases and to a lesser extent cellulases have been proven most effec- Various kinds of NSP-degrading enzymes have been reported in tive in broilers (Leslie, Moran, & Bedford, 2007). Mannanases and animal feed, which include cellulase, hemicellulase, xylanase, pec- pectinases have shown good results in a soy-based diet (Jackson, tinase, b-glucanase and a-galactosidase. Their effects and benefits Geronian, Knox, McNab, & McCartney, 2004). However, in a corn- are illustrated in Table 5. b-Glucanases and xylanases have been soy-based diet xylanase and glucanase were more effective in successfully used in monogastric diets to hydrolyse NSPs, such as breaking down the insoluble fibre fraction. Moreover, Li, Sauer, barley b-glucans and arabinoxylans (Cowan, 1996). Moreover Yin, and Huang (1996) suggested that the improvement in digestibility Baidoo, Schulze, and Simmins (2001) reported that inclusion of of nutrients in barley-based diets with cellulase addition for young b-glucanase (600 units/kg diet) and xylanase (745 units/kg diet) pigs was due to the increased degradation of b-linked components in pig feeds containing hull-less barley decreased the viscosity in in barley, which made them available to the host animal. the distal part of the small intestine, lowered the plasma urea nitrogen concentration and increased the apparent ileal digestibil- 12.1.2. Reduction of digesta viscosity ity of energy and some amino acids. A similar beneficial effect of Studies on monogastric animals have shown that reduced NSP-degrading enzymes in pigs has been reported by Yin et al. digesta viscosity due to NSP-degrading enzyme supplementation (2000). Besides, addition of NSP-degrading enzymes during feed is the main factor responsible for the observed enhanced perfor- production was found to degrade NSPs and markedly improved mance response on feeding plant materials rich in NSPs (Cowieson, the digestion and absorption of feed components as well as growth Singh, & Adeola, 2006). However, Partridge (2001) demonstrated performance in broiler chickens (Choct, Hughes, Trimble, Angka- that pig small intestinal viscosity is relevant for its performance naporn, & Annison, 1995; Ghorbani, Fayazi, & Chaji, 2009). Almirall but it is of a lower order of importance than for broilers. To our et al. (1995) showed that broiler chicks fed with high viscosity bar- knowledge no study is available which illustrates the effect of sup- ley had lower amylase and lipase activities in the digesta, while plementing NSP-degrading enzymes on the intestinal viscosity in addition of b-glucanase in the diet increased activities of the two fish fed NSP-containing diets. enzymes and of trypsin as well. This observation was consistent with the result of Li, Li, and Wu (2009) with tilapia (Oreochromis 12.1.3. Stimulation of bacterial population niloticus Â Oreochromis aureus) where 1 g/kg of NSP-degrading Addition of NSP-degrading enzymes in feed breaks down plant enzyme (50 FBG (fungal b-glucanase units)/g; Roche Shanghai) in- cell wall carbohydrates and reduces chain length, producing smal- creased activity of amylase in the hepatopancreas and intestine by ler polymers and oligomers. These fragments further become small 11.4% and 49.5%, respectively. The application of Natugrain-blendÒ enough to act as a substrate for bacterial fermentation that can be (containing b-glucanase and b-xylanase) at concentrations of 75, beneficial with VFA production and altering the bacterial popula- 150 and 300 ll/kg in diets containing 30% wheat or dehulled lupin tion. Several studies have shown that applications of exogenous had no remarkable effect on dry matter, energy or protein digest- enzymes significantly alter VFA production and the population ibilities when fed to silver perch (Stone, Allan, & Anderson, profiles of gut-associated microflora (Bedford & Apajalahti, 2001). 2003a). According to Stone et al. (2003a) the missing effect of Nat- Yin et al. (2000) studied the effects of xylanase supplementation ugrain-blendÒ inclusion on nutrient digestibility could be due to in a wheat-bran-based diet in young pigs, and found that the en- the intolerance of silver perch to high levels of galactose and xylose zyme addition increased the ileal production of VFA. in their blood (Stone, Allan, & Anderson, 2003b). Prolonged eleva- However, care must be taken while selecting the feed enzyme tion of blood galactose has been reported to reduce growth perfor- because some products can be overdosed and reduce the size of mance in carp (Shikata, Iwanaga, & Shimego, 1994). the oligosaccharides down too far, to monosaccharides. If excess The pretreatment of dietary plant materials with exogenous monosaccharides are produced, it may result in osmotic diarrhoea carbohydrases (a-amylase, b-glucanases and b-xylanases) en- and/or poor performance (Schutte, 1990). hances the energy digestibility by releasing more glucose, galact- Currently, the use of various exogenous feed enzymes is consid- ose and xylose (Kumar et al., 2006). The supplementation of two ered to reduce FM inclusion by around 5% in most aquafeeds and NSP-degrading enzymes, for example 400 mg ‘VP’ (contains mainly there is a potential to reduce further the demand for FM by the glucanase, pentosanase and cellulase, each at 50 IU per g) and aquaculture sector in the coming years (Felix & Selvaraj, 2004). 800 mg ‘WX’ (contains mainly xylanase, 1000 IU per g) in basal However, lack of information on the nutritive value and the con- feed of Japanese seabass (Lateolabrax japonicus) significantly en- tent and nature of anti-nutrients in feed ingredients make difficult hanced the specific growth rate, feed efficiency ratio, nitrogen the selection of suitable enzymes or enzyme mixtures as feed addi- retention and reduced ammonia excretion (Ai et al., 2007). tives. Further studies are required in this area. Another constraint is that feed enzymes are proteins, which are substrate specific, 12.1. Mechanism responsible for enzymic degradation of non-starch work within a particular pH range and are themselves potentially polysaccharides open to digestion by endogenous enzymes. In the stomach pH is low and suited primarily to protein digestion (Yin et al., 2001). The mechanisms by which exogenous NSP-degrading enzymes Consequently it is crucial that enzymes are protected to a sufficient enhance nutrient digestion and utilisation from plant proteins in extent against proteolysis and are available at the targeted site of fish have not yet fully understood. However, based on various re- activity (Yin et al., 2001). A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1423

13. Conclusions Asp, N. G., Johansson, C. G., Hallmer, H., & Siljeström, M. (1983). Rapid enzymatic assay of insoluble and soluble dietary fibre. Journal of Agricultural and Food Chemistry, 31, 476–482. In aquaculture the use of plant-based protein is increasing at a Asp, N. G., Schweizer, T. F., Southgate, D. A. T., & Theander, O. (1992). Dietary fibre fast pace due to its relatively low cost and ample availability. Con- analysis. In T. F. Schweizer & C. A. Edwards (Eds.), Dietary fibre-a component of sequently, the use of non-starch polysaccharides (NSPs), a class of food. Nutritional function in health and disease (pp. 57–102). London: Springer- Verlag. anti-nutrients present in plant-based diets, will increase in the fu- Bach Knudsen, K. E. (1997). Carbohydrate and lignin contents of plant materials ture. Currently there is relatively little information on the effects of used in animal feeding. 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