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, Belgiumb Institute for Animal Production in the Tropics and Subtropics, University of Hohenheim, 70599 Stuttgart, Germany

a r t i c l e i n f o

Article history:Received 22 November 2010Received in revised form 28 January 2011Accepted 8 February 2011Available online 12 February 2011

Keywords:Fish feedNon-starch polysaacharideAnti-nutritive effectsNutrient metabolismImmunostimulants

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.02.042

⇑ Corresponding author. Tel.: +49 711 45923640; faE-mail address: makkar@uni-hohenheim.de (H.P.S

1 These authors contributed equally to this review.

a b s t r a c t

The success and sustainability of aquaculture depends on minimising the operational cost of feed that ingeneral comprises 50–60% of the total cost in intensive farming. The major feed ingredient, fish meal, isexpensive and there is increasing competition with other livestock industries for the available static sup-ply of fish meal. Hence, the incorporation of plant-derived materials in fish feeds is receiving increasingattention. One of the main constraints in the utilisation of plant ingredients in aquaculture is the presenceof indigestible carbohydrates, which consist primarily of non-starch polysaccharides (NSPs). These form apart of the cell wall structure of cereals and legumes. The presence of NSPs in the diet interferes with feedutilisation and adversely affects performance of the animal. Supplementation of NSP-degrading enzymesin feed mitigates the adverse effects of NSPs. The effects of NSPs in pigs and poultry have been widelystudied; however little information exists for fish. This review synthesizes the available information onfish and highlights the knowledge gaps. It is hoped that this review will provide a momentum to theresearch on the roles of NSPs in fish nutrition and physiology and on the efficient use of NSP-degradingenzymes.

� 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14102. Classification of non-starch polysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1410

2.1. Cellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14112.2. Non-cellulosic polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412

2.2.1. Arabinoxylans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14122.2.2. Mixed-linked b-glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14122.2.3. Mannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412

2.3. Pectic polysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1413

2.3.1. Arabinans, galactans and arabinogalactans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1413

3. Non-starch polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14144. Non-starch polysaccharides in fish feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14145. Methods for non-starch polysaccharides quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1415

5.1. Gravimetric analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14155.2. Monomeric component analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1416

6. The anti-nutritive effect of non-starch polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1416

6.1. Modulation in digesta viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14166.2. Alteration in gastric emptying and rate of passage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14176.3. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14187.2. Effect on protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14187.3. Effect on lipid and cholesterol level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14197.4. Effect on minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1419

ll rights reserved.

x: +49 711 45923702.. Makkar).

1410 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426

8. Effect on growth performance and body composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14199. Non-starch polysaccharides and effect on gelatinisation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142010. Interaction with antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142011. Purified non-starch polysaccharides as immunostimulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142012. 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142212.1.2. Reduction of digesta viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142212.1.3. Stimulation of bacterial population. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422

13. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1423References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1423

1. Introduction

The growing demand for fish and limited supply from wild cap-ture are giving momentum to the development of aquaculture. Theprogress of culture-based fisheries is determined mainly by thequality of feed delivered. Fish meal (FM) having high protein con-tent and favourable amino acid profile is highly preferred by fishculturists. The total world FM production is about 5–6 million ton-nes per annum, which accounts for 4–5% of total fish production of144 million metric tonnes (Food, 2008). In spite of being the mostimportant protein source in commercial feeds, production of FM isrestricted to certain parts of the world only; as a result it is becom-ing too expensive for many aquaculture practising countries. Fur-thermore, the abundance of FM appears to be ending, since thelevel of FM production is expected to remain stable over the next10 years (Mazurkiewicz, 2009). As a consequence, the fisheries sec-tor may have to undergo a recessionary phase over the comingyears. In order to provide sustainability, therefore, it is of utmostimportance to reduce the presence of FM in aquafeeds and replaceit with plant-based sources. The higher availability and low cost ofplant-based feeds give them advantages over FM. Although the car-bohydrate component of grains and legumes may provide a cheapsource of dietary energy for fish, it is poorly utilised, compared toprotein and lipid, by most fish species (Allan et al., 2000). In addi-tion, the quality and level of protein in, and palatability of, plant-based protein sources are generally inferior to FM. However, themain limitation with plant-derived materials, such as legumeseeds, soybean meal, different types of oilseed cake, canola (rape-seed) meal, sunflower oil cake, root tuber meal, is the presenceof a wide range of anti-nutritional factors, such as protease inhib-itors, non-digestible carbohydrates, lectins, saponins, phytates andpossibly allergenic storage proteins (Francis, Makkar, & Becker,2001). In addition to these factors that hamper digestion in fish(Refstie, Svihus, Shearer, & Storebakken, 1999; Storebakken,Shearer, & Roem, 1998), non-starch polysaccharides (NSPs) playan important role. In general, NSPs are a complex group, composedpredominantly of linked monomers of hexoses and pentoses, e.g.,galactose, glucose, arabinose, xylose and mannose (van Barneveld,1999). The NSP content in wheat and lupin may account for 25%and 50% of the total grain and seed respectively and acts as the pri-mary energy storage carbohydrate in lupin (van Barneveld, 1999).However, in fish and other monogastric animals enzymes such asb-glucanases or b-xylanases that digest NSPs are scarce or non-existent (Kuz’mina, 1996). Consequently, the dietary NSPs remainindigestible and cannot be used as an energy source. The additionof NSP-containing feedstuffs to the diets of monogastric animals,for example, broiler and swine, reduces the apparent digestibilityof the diet and has negative impacts on growth. However, only alimited number of such studies have been conducted in fish. Refstieet al. (1999) have demonstrated negative effects of NSPs on diges-tion and absorption of lipid in Atlantic salmon. Non-starch polysac-

charides are also thought to be responsible for a slower rate ofgastro-intestinal passage of NSP-containing diets in fish (Storebak-ken, Kvien, Shearer, Grisdale-Helland, & Helland, 1999). Feeding ofsalmonids with diets incorporating NSP has been shown to reducethe availability of nutrients (Storebakken & Austreng, 1987).

At present approximately 2.0 billion tonnes of cereal grainsand 140 million metric tonnes of legumes and oil seeds are pro-duced worldwide per year and approximately 230 million metrictonnes of fibrous materials are produced as a by-product. Thiswide availability of plant resources can very well be utilised ascheaper fish feed ingredients, through proper management ofthe NSPs in these plant materials. Feed processing and utilisationof exogenous enzymes (b-glucanase and b-xylanases) have beenused to decrease the negative effects of NSP and thus to improvethe nutritive value of feed. Moreover, there is a contemporarytrend to seek feed ingredients which may contribute to betterhealth by interfering with colonisation and microbial growth inthe gut. In this regards NSPs such as b-glucans and mannose havebeen shown to have immunostimulating activities (Kumar, Sau-rabh, Sahu, & Pal, 2005).

2. Classification of non-starch polysaccharide

The term NSP covers a large variety of polysaccharide mole-cules, excluding a-glucans (starch). NSPs have been classifiedbased on different criteria. Historically, the classification wasbased originally on the methodology used for extraction andisolation of polysaccharides. The residue remaining after a seriesof alkaline extractions of cell wall materials was called cellulose,and the fraction of this residue solubilised by alkali was namedhemicellulose (Neukom, 1976). Another classification was basedon the differences in solubility. This classification includes threecategories of NSP, namely crude fibre (CF), neutral detergent fi-bre (NDF) and acid detergent fibre (ADF). CF refers to theremnants of plant material after extraction with acid and alkali,and includes variable portions of insoluble NSP. NDF comprisesthe insoluble portion of NSP plus lignin, while ADF refers to aportion of insoluble NSP comprised largely, but not solely, ofcellulose and lignin. However, this basis of categorisation lackedprecision with respect to both chemical structures and biologicalfunctions and, moreover, the nutritional significance of val-ues obtained using this method in monogastric nutrition isdoubtful.

Bailey (1973) proposed a clearer classification of NSP into threemain groups, namely cellulose, non-cellulosic polymers and pecticpolysaccharides. Arabinoxylans, mixed-linked b-glucans, mannans,and xyloglucan come under the category of non-cellulosic poly-mers while polygalacturonic acids substituted with arabinan,galactan and arabinogalactan are included in the group of pecticpolysaccharides (Table 1).

Table 1Classification of non-starch polysaccharides.

Category Monomeric residue Linkage Sources

Cellulose Glucose b-(1 ? 4) Most cereals and legumes

Non-cellulosic polymersArabinoxylans Arabinose and Xylose b-(1 ? 4)-linked xylose units Wheat, rye, barley, oat, rice,

sorghumMixed-linked b-

glucansGlucose b-(1 ? 3) and b-(1 ? 4) Oat and barley

Mannans Mannose b-(1 ? 4) Coffee seedGalactomannans Galactose and

mannansb-(1 ? 4)-linking mannan chains with a-(1 ? 6)-linked galactosyl side groups Locust bean gum and guar gum

Glucomannans Glucose and mannans b-(1 ? 4)-linked mannan chain with interspersed glucose residues in the mainchain

Sugar-beet pulp, lilies, irises

Pectic polysaccharidesArabinans Arabinose a-(1 ? 5) Cereal co-productsGalactans Galactose b-(1 ? 4) Sugar bean meal, sugar-beet pulpArabinogalactans (Type

I)Arabinose andGalactose

b-(1 ? 4) galactan backbone substituted with 5-linked and terminal arabinoseresidues

Grain legumes

Arabinogalactans (TypeII)

Arabinose andGalactose

b-(1 ? 3,6)-linked galactose polymers associated with 3- or 5-linked arabinoseresidue

Rapeseed cotyledon

Fig. 1. Chemical structure of cellulose.

A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1411

2.1. Cellulose

Cellulose is a complex polysaccharide, consisting of 3000 ormore b-(1 ? 4) linked D-glucose units. This bond generally makescellulose ingestible for monogastric animals, due to lack of the en-zyme cellulase in the digestive tract. Cellulose is the basic struc-tural component of plant cell walls and comprises about 33% ofall vegetable materials. It is the most abundant of all naturally-occurring organic compounds, comprising over 50% of all the car-bon in vegetation.

Cellulose quantity in whole grains can vary from species to spe-cies and is largely a consequence of the thickness of the husk andseed coat. Cells which contain more cellulose tend to have thickerand stronger cell walls. Seed endosperm cells have only thin cellwalls and in a well-filled grain the proportion of cellulose to starch,

Fig. 2. Chemical structu

or other reserve polysaccharide, should be low (Brett & Waldron,1996).

Cellulose is a straight-chain polymer where no coiling orbranching occurs, and the molecule adopts an extended and ratherstiff rod-like conformation, aided by the equatorial conformation ofthe glucose residues. The multiple hydroxyl groups on the glucoseresidues from one chain form hydrogen bonds with oxygen mole-cules on the same or on a neighbouring chain, holding the chainsfirmly together side-by-side (Fig. 1). The chains can stack togetherto form larger microfibrils which make cellulose highly insoluble inwater, but can swell in concentrated sodium hydroxide solutions.Through the use of hydrogen-bond breaking reagents, such as N-methylmorpholine N-oxide, cellulose can be brought into solution.Moreover, to be used as a dietary supplement, cellulose-rich maizebran can be converted to a cellulosic gel through thermal and sheartreatments, followed by alkaline peroxidation and shearing(Fincher & Stone, 2004). Cellulose microfibrils may also associatewith water and matrix polysaccharides, such as the (1 ? 3,1 ? 4)-b-D-glucans, heteroxylans (arabinoxylans) and glucomann-ans (Fincher & Stone, 1986a).

The hydrolysis of cellulose is limited in animals but can be occurin some microbes, such as bacteria and fungi (Xiao & Xu, 2002).Since fish lack cellulase in their intestines, cellulose is indigestiblein fish; and therefore it is of no nutritional value in formulated fishfeeds. However, developments in fish nutrition and aquaculture

re of arabinoxylan.

1412 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426

technology have encouraged the use of cheaper feed ingredients;including those containing cellulose. Therefore, it is important todevelop methods for the improvement of enzymatic hydrolysis ofcellulose.

2.2. Non-cellulosic polymers

2.2.1. ArabinoxylansArabinoxylans have been identified in a variety of tissues of the

main cereals: wheat, rye, barley, oat, rice, sorghum (Fincher &Stone, 1986b). Although these polysaccharides are minor compo-nents of entire cereal grains, they constitute an important part ofplant cell walls. Thin walls that surround the cells in the starchyendosperm and the aleurone layer in most cereals consist predom-inantly of arabinoxylans (60–70%); exceptions are endosperm cellwalls of barley (20%) and rice (40%) (Fincher & Stone, 1986b).Non-endospermic tissues of wheat, particularly the pericarp andtesta, also have very high arabinoxylan content (64%) (Selvendran& DuPont, 1980).

The structures of cereal arabinoxylans are composed predomi-nantlyoftwopentoses,arabinoseandxylose(Izydorczyk&Biliaderis,1995). Their molecular structure consists of a linear backbone of b-(1 ? 4)-linked xylose units to which substituents are attachedthrough O-2 and O-3 atoms of the xylosyl residues (Perlin, 1951)(Fig. 2). Arabinoxylans form highly viscous aqueous solutions whichmay create problems when wheat is used in processes, such asproduction of grain spirits. The degree of arabinose substitutions willinfluence the conformation adopted by arabinoxylans and the result-ing viscosity of solutions.

When the arabinose residues are stripped off the xylan back-bone (using oxalic acid), aggregation appears at a xylose-to-arabi-nose ratio of about four and precipitation occurs when this isincreased above ten (Sternemalm, Höije, & Gatenholm, 2008).The loss of arabinose side chains also correlates with a loss inwater-binding capacity (Sternemalm et al., 2008). When this is ab-sent the molecule binds less water and becomes less soluble. Thedegree of arabinose substitution has little influence on the overallsemi-flexible conformation and hence the viscosity (Sternemalmet al., 2008). However, in wheat flour, the distribution of the typeof substitution is not random but the distribution of substituted(irrespective of of the substitution type) residues along the chainappears random (Dervilly-Pinel, Tran, & Saulnier, 2004). The arab-inose residues may also be linked to other groups attached, such asglucuronic acid residues, ferulic acid crosslinks and acetyl groups(Sørensen, Pedersen, & Meyer, 2007).

Most of the arabinoxylans in cereal grains are insoluble in waterbecause they are anchored in the cell walls by alkali-labile ester-like cross links (Mares & Stone, 1973). But the arabinoxylans notbound to the cell walls can form highly viscous solutions and theycan absorb about ten times their weight of water. In the presenceof oxidative agents, such as H2O2/peroxidase, arabinoxylans canrapidly develop a gel network, as a result of the re-establishmentof cross-links (Geissmann & Neukom, 1973). Fully cross-linked ara-binoxylans hold up to 100 g of water per g polymer (Izydorczyk &Biliaderis, 1995).

Fig. 3. Primary structure of b-D-g

Apart from covalent cross-links, arabinoxylans may also form‘‘junction zones’’ by intermolecular hydrogen bonding betweenunsubstituted regions of the xylan backbone (Fincher & Stone,1986b). Not only can arabinoxylans establish covalent cross-links,they may also form ‘‘junction zones’’ by intermolecular hydrogenbonding between unsubstituted regions of the xylan backbone(Fincher & Stone, 1986b; Sørensen et al., 2007). Such type of inter-action of arabinoxylans may be of great importance in determiningtheir conformational changes and solubility properties, and thustheir anti-nutritional activities.

2.2.2. Mixed-linked b-glucansThe physical and physiological properties of b-glucans are of

commercial and nutritional importance. Increasing interest in b-glu-cans during the last two decades is largely due to their acceptance asfunctional, bio-active ingredients (Cui & Wood, 2000). Cereal b-glu-cans have been associated with the reduction of plasma cholesteroland a better control of postprandial serum glucose levels in humansand animals (Bhatty, 1999). The mixed-linked b-glucans are uniqueto the Poales, the taxonomic order that includes cereal grasses. Theyare also known as cereal b-glucans and are located in the subaleu-rone and endospermic cell wall (Ebringerová, 2006) where theyassociate with cellulose microfibrils during cell growth. Oats andbarley contain 3–12% and more depending on the cultivar.

The ubiquitous structural features of these polysaccharides arewell established. The structural features of b-glucans are importantdeterminants of their physical properties and functionality, includ-ing their physiological responses when they are considered asingredients in cereal-based foods and other formulated products.These features include ratios of linkages joining glucose units,presence and amount of long cellulose-like fragments, and molec-ular size (Izydorczyk & Biliaderis, 2000). In general, b-glucans con-sists of a linear chain of glucose units joined by both b-(1 ? 3) andb-(1 ? 4) linkages (Bengtsson, Åman, & Graham, 1990) (Fig. 3).

It should be noted that the mixed-linked b-glucans and cellu-lose are both comprised of b-linked-glucose units but there is littlesimilarity in their physical properties. Cellulose consists only of(1 ? 4)-b-linkages and is therefore stiff, highly crystalline andnon-soluble. On the other hand, the b-(1 ? 3)-linkages break upthe regular structure of b-(1 ? 4) chains of the b-glucan moleculeand make it soluble and flexible (Anderson & Bridges, 1993). There-fore, the b-glucans are often isolated by aqueous extraction fol-lowed by precipitation with ammonium sulphate (McCleary,1986).

The potential application of b-glucans as food hydrocolloids hasbeen also proposed. In addition, b-glucans have been shown toform gels under certain conditions (Lazaridou, Biliaderis, &Izydorczyk, 2003) and can be utilised as thickening agents to mod-ify the texture and appearance in gravies, salad dressings and icecream formulations (Wood, 1986) or may be used as fat mimeticsin the development of calorie-reduced foods (Inglett, 1990).

2.2.3. MannansLinear chains of b-(1 ? 4) mannan are found in the cell walls of

the coffee seed endosperm (Wolfrom, Layer, & Patin, 1961). In most

lucans (Ebringerová, 2006).

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

Fig. 5. Primary structure of glucomannans (Ebringerová, 2006).

A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1413

cases, these polysaccharides are highly insoluble in water and verydense. Accordingly, it has been suggested that mannans form themolecular basis for the hardness of the plant. Mannan has alsobeen reported to be present in the cell walls of several siphona-ceous green algae in the families Acetabularia, Codium andHalicoryne (Frei & Preston, 1968). Furthermore, mannan is alsofound in some red algae, such as Porphyra umbilicalis (Jones,1950). In some of these algae, mannan is the main structural poly-mer and displays microfibrillar morphology (Chanzy, Grosrenaud,Vuong, & Mackie, 1984).

The mannan-type hemicelluloses, can be divided into twogroups: (i) galactomannans and (ii) glucomannans (Ebringerová,2006).

2.2.3.1. Galactomannans. Galactomannans are reserve polysaccha-rides in the seed endosperm of leguminous plants (Leguminosae).They are water-soluble and can imbibe water, thus providing awater-holding function for the seed (Reid, 1985). They are com-posed of b-(1 ? 4)-linked mannan chains with a-(1 ? 6)-linkedgalactosyl side-groups (McCleary, 1985) (Fig. 4). Both the solubilityand the viscosity of the galactomannans are influenced by themannose-to-galactose ratio, which can vary from 1 to 5 (Reid,1985). Furthermore, the distribution of the substituents can varyconsiderably, which also affects the physical properties of galacto-mannans (Daas, Schols, & de Jongh, 2000). Two of the most wellcharacterised galactomannans are found in locust bean gum andguar gum, isolated from the seeds of Ceratonia siliqua and Cyanap-osis tetragonolobus, respectively (Rol, 1973).

2.2.3.2. Glucomannans. Glucomannans are present as a minor com-ponent in cereal grains (Fincher & Stone, 1986b) and act as storagepolysaccharides in the seeds of certain annual plants, for examplesome lilies (Liliaceae) and irises (Iridaceae) (Meier & Reid, 1982).Glucomannans are also found in the bulbs, roots and tubers of sev-eral other types of plants. Many of these glucomannans are water-soluble and are composed of a b-(1 ? 4)-linked mannan chain withinterspersed glucose residues in the main chain; they are oftenacetylated (Fig. 5). The mannose-to-glucose ratio ranges from 4:1to less than 1:1 (Meier & Reid, 1982). The structural and physico-

chemical properties of the storage glucomannan, known as ‘konjacmannan’ in the food industry, have been reinvestigated (Nishinari,Williams, & Phillips, 1992). The results indicate that this polysac-charide, composed of glucose and mannose in the ratio 1–1.6,has the backbone slightly O-acetylated, and branched (about 8%)at position 6 of the glucopyranose residues. The branches termi-nated by both glucopyranose and mannopyranose have been sug-gested as a new structural feature.

2.3. Pectic polysaccharides

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) glycosidicinkage. These uronic acids have carboxyl groups, which in the pres-ence of divalent cations (usually calcium) have considerable effectson viscosity, solubility, and gelation formation (Thakur et al.,1997).

2.3.1. Arabinans, galactans and arabinogalactansThese three classes of polymers together constitute the neutral

pectic substances. Pure arabinans and galactans are present inplant cell wall but in a very low amount. The arabinans are poly-mers of (1 ? 5)-a-L-arabinose residues with some degree ofbranching through O-2, O-3 or both positions. Galactans are b-1,4linked linear polymers, which can possess a small number of 6-linked residues (Ghosh & Das, 1984).

The arabinogalactans occur in two distinct types in plant cellwalls. Type I, which is very common in grain legumes, is character-ised by b-(1 ? 4) galactan backbone substituted with 5-linked andterminal arabinose residues (Cheetham, Cheung, & Evans, 1993).The type II arabinogalactan is commonly found in rapeseed cotyle-don (Siddiqui & Wood, 1972). Type II is characterised by b-(1 ? 3,6)-linked galactose polymers associated with 3- or 5-linkedarabinose residue. Unlike type I arabinogalactans, type II are not astructural component of the cell wall but are thought to be associ-ated with extracellular space and with plasmalemma. However, alow molecular weight type II arabinogalactan associated with a

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

Ingredients Cellulose Total NSP Lignin Soluble NSPa Insoluble NSP

CerealsMaize 22 97 11 9 66Wheat 20 119 19 25 74Wheat bran 72 374 75 29 273Rye 16 152 21 42 94Barley: hulled 43 187 35 56 88Barley: hull-less 10 124 9 50 64Corn gluten 75 351 – 34 242Oats: hulled 82 232 66 40 110Oats: hull-less 14 116 32 54 49Oat hull meal 196 505 148 13 295

Legumes/Seed mealPeas 53 180 12 52 76Soybean meal 62 217 16 63 92Rapeseed meal 52 220 134 55 123Lupins 131 405 – 134 139Sunflower cake 123 315 – 57 136Jatropha 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 – – –

MiscellaneousSugar-beet pulp 195 779 35 407 177Cottonseed cake 92 257 – 61 103Cottonseed meal 90 283 – 66 127Alfalfa leaf meal 139 329 _ 77 113

a Non-cellulosic polysaccharides.b Our unpublished data.

1414 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426

hydroxyproline-rich peptide (arabinogalactan proteins, AGPs) hasalso been isolated from wheat flour (Fincher & Stone, 1974).

3. Non-starch polysaccharides

Non-starch polysaccharides are complex polysaccharides otherthan the starch. These polysaccharides are typically long polymericcarbohydrate chains containing up to several hundred thousandmonomeric units. Non-starch polysaccharides can comprise up to90% of the cell wall of plants (Selvendran & DuPont, 1980). Themost abundant plant cell wall NSPs include cellulose, hemicellu-lose and pectins; while fructans, glucomannans and galactomann-ans belong to the group of NSP that is not so abundant as cellulose,hemicellulose or pectins and serves as the storage polysaccharideswithin the plants. Mucilages, alginates, exudate gums, b-glucansand various modified polysaccharides are other constituents ofthe non-starch polysaccharides (Asp, Schweizer, Southgate, &Theander, 1992). The physiological impact of individual NSPs isdependent on the sugar residues present and nature of the linkagepresent between these residues. However, NSPs differ from starchnot only in the type of monomers present but also differ by thenumber and type of monomeric units linked together, the orderin the chain and the types of linkages between the various mono-mers. Starch is composed entirely of glucose monomers, which arelinked by a-glycosidic bonds while NSPs are composed of differentkinds of monomers, which are linked predominantly by b-glyco-sidic bond. The difference in bonding structure has profound ef-fects on digestibility, as different classes of enzymes are requiredfor the hydrolysis of a- and b-glycosidic bond. The predominantstarch digestive enzymes are a-amylase (1,4-a-D-glucan glucano-hydrolase), a-glucosidase (1,4-a-glucosidase) and oligo-1-6-gluco-sidase. In com bination, these enzymes specifically hydrolyse thea-glycoside bonds of starch to yield glucose. The activity of theseenzymes in fish intestine varies with fish species, and is based ontheir feeding habits. Activity of these enzymes is high in herbivo-rous, medium in omnivorous and low in carnivorous fish (Krog-dahl, Hemre, & Mommsen, 2005; Kuz’mina, 1996). On the other

hand, the enzymes required to digest NSP, such as b-glucanaseand b-xylanases, are very scarce or even absent among fish species(Kuz’mina, 1996).

Apart from NSPs, other unavailable carbohydrates in food areoligosaccharides, fructans, lignin and resistant starch. However,NSPs along with lignin and resistant starch are the major constitu-ents of total dietary fibre. Lignin is a high-molecular-weight poly-mer composed of phenylpropane residues, formed by thecondensation of the aromatic alcohols, cinnamyl, guaiacyl andsyringyl alcohols (Southgate, 1993). Resistant starch can be definedas that fraction of dietary starch, which escapes digestion in thesmall intestine. It is the sum of starch and degradation productsof starch.

4. Non-starch polysaccharides in fish feed

The NSPs in aquaculture feeds are present as an integrated partof the cell wall of plant ingredients and also in a purified solubleform, such as guar gum, to stabilise the pellet. In general NSPsare present in two wide categories of crops, namely cereal grainsand legumes. Non-starch polysaccharides in cereal grains are com-posed predominantly of b-glucans, arabinoxylans, and cellulose.Wheat, rye and triticale contain substantial amounts of both solu-ble and insoluble NSP, while very low levels of NSPs are present incorn and sorghum. Grain legumes also contain considerableamounts of NSPs. Pectic polysaccharides are the main NSPs presentin the cotyledon of legumes. Cellulose and xylans, besides beingthe major NSPs in cereal grains, are only found in the hulls or husksof most legumes.

Apart from cereals and legumes, NSPs are also present in otherplant parts, such as roots, tubers and leaves. These ingredients areused in many countries as possible feed resources. The NSP contentof ingredients used as aqua feeds is presented in Table 2. Moreover,an extensive report on carbohydrate and lignin contents of plantmaterials used in animal feeding has already been published byBach Knudsen (1997).

A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1415

5. Methods for non-starch polysaccharides quantification

It is difficult to develop an accurate analytical method for esti-mating NSP content in feed. This is because of the complexity anddiversity of the polysaccharides involved. In general gravimetricand monomeric component analytical approaches are used forquantifying NSPs. The gravimetric method of fibre analysisassumes that all residues are present in fibre, whereas componentanalysis quantifies the amount of constituent sugars present in asubstrate and then, via summation, determines the total NSPconcentration.

5.1. Gravimetric analysis

Gravimetric method is the traditional way of fibre analysis,which involves chemical or enzymatic solubilisation of dietary pro-tein, starch and fat, followed by weighing of the insoluble residue.The crude fibre estimation is an example of gravimetric analysis.

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

Factors Effects Refe

Changes in digestaviscosity

� Reduced mixing of digestive enzymes andsubstrates� Hindered effective interaction of digestive

enzyme at the intestinal mucosal surface� 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

ChoIkegAmiLeen

Alteration in the gastricemptying and rate ofpassage

� Reduced rate of gastric emptying� Increased rate of passage of stomach content� Delayed intestinal absorption of glucose� Reduced plasma cholesterol and glucose

levels

RainPotk(199Leen

Alteration of gutphysiology

� Hinder endogenous secretion of water, pro-teins, electrolytes and lipids� Enhanced bile acid secretion, and significant

loss of these acids in the faeces� Hampered absorption of lipids and choles-

terol in intestine� Limited intestinal enzyme activity

PettAngChoHos

Alteration in the gutmorphology

� Increased size and length of digestive organs� Reduced concentrations of DNA in jejunum,

ileum, and liver, indicating programmed celldeath� Augmented concentrations of RNA in the

colon� Reduced villi length� Increased depth of intestinal crypts in jeju-

num and ileum� Impaired water absorption, can lead to

dehydration� Increased rate of turnover of intestinal muco-

sal cells

BaseLeen

Alteration in the native gutmicroflora

� Stimulated microbial fermentation inintestine.� 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 ingut� Influenced bioavailability of dietary minerals� Decreased oxygen tension, favouring devel-

opment of anaerobic microbiota

WooLeen

Alteration in gut mucuslayer

� Increased concentrations of lumenal mucin instomach and small intestine

Satc

However, it is not an accurate estimation of total NSPs, since therecovery of cellulose, hemicelluloses and lignin is low (Van Soest& McQueen, 1973). The refined form of gravimetric crude fibreanalysis is the detergent method of fibre analysis, which delineatesthe form of fibre present in feedstuffs. The two forms of detergentfibre are neutral detergent fibre (includes cellulose, hemicelluloseand lignin) and acid detergent fibre (includes cellulose and lignin),the difference of these two fractions is an estimate of hemicellulosein a feed. Even though detergent methods of fibre analysis havemany advantages over crude fibre estimation, both underestimatethe amount of total fibre in a feed, due to inability to recover pec-tins, mucilages, gums and b-glucans which are soluble componentsof fibre. Asp, Johansson, Hallmer, and Siljeström (1983) developedan enzyme-based gravimetric method, in which a sample is pre-treated with enzymes for the digestion of starch and protein,followed by the recovery of soluble components via precipitationin ethanol and the insoluble components by filtration. This methodwas further modified by Jeraci, Lewis, Van Soest, and Robertson(1989), incorporating a urea enzymatic dialysis to assure the

rences

ct et al. (1996);ami et al. (1990); Hossain et al. (2001);rkolaie, Leenhouwers, Verreth, and Schrama (2005);houwers et al. (2007a, 2007b)

bird and Low (1986);ins, Lawrence, and Thomlinson (1991); Shimeno et al. (1992); Kaushik et al.5); Refstie et al. (1999); Bach Knudsen (2001); Hossain et al. (2001);houwers et al. (2007a, 2007b)

ersson and Åman (1989);kanaporn et al. (1994);ct (1997);sain et al. (2001)

rga, (1985); Jin et al. (1994); Nabuurs (1998); McDonald (2001); Iji et al. (2001);houwers et al. (2006)

d and Serfaty-Lacrosniere (1992); Choct (1997); Amirkolaie et al. (2006);houwers et al. (2007a, 2007b)

hithanandam, Klurfeld, Calvert, and Cassidy (1996)

1416 A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426

removal of essentially all starch. Moreover, during the same decadeTheander and Åman (1982) developed an indirect method toanalyse total dietary fibre (TDF) in food stuff by quantifying theamounts of uronic acids, sugars, Klason lignin and starch and thencalculating the TDF as the sum of the uronic acids, sugarsand Klason lignin, minus the concentration of starch. In contrast,Prosky et al. (1984) developed a direct method of TDF quanti-fication. These assays have been further expanded to allowquantification of both soluble and insoluble dietary fibre compo-nents (Theander, Åman, Westerlund, Anderson, & Pettersson,1995) and refined to increase precision and decrease the complex-ity and time required (Lee, Vincet, Prosky, & Sullivan, 1996).

5.2. Monomeric component analysis

Monomeric component analysis was first developed by Englystand Cummings (1988). In this method all starch is hydrolysedenzymatically and NSPs are measured as the sum of the constitu-ent sugars released by acid hydrolysis. The individual sugars aresubsequently quantified by gas chromatography (GC) or byhigh-performance liquid chromatography (HPLC) (Englyst, King-man, & Cummings, 1992; Englyst, Quigley, & Hudson, 1994).Moreover, a single value for total sugars may be obtained by a col-orimetric procedure that measures NSPs as reducing sugars (Eng-lyst et al., 1994). The GC technique for dietary fibre analysis,preferred by many researchers, measures NSP as the sum of neu-tral sugars obtained by GC and uronic acids measured separately(Mongeau et al., 2001). In this procedure, the sugars are reducedto their alditols with alkaline sodium borohydride and acetylatedwith acetic anhydride in the presence of methylimidazole as cat-alyst. Since the uronic acid-containing polysaccharides are moredifficult to hydrolyse and require treatment with concentratedacid at high temperature; they are measured separately by color-imetry (Scott, 1979). The HPLC method of dietary fibre analysis(Englyst et al., 1994) is very common and measures NSPs as thesum of neutral sugars and uronic acids, directly by electrochemi-cal detection.

It must be noted that the values obtained by GC or HPLC aretypically lower in comparison to the gravimetric method of analy-sis because of the exclusion of lignin and resistant starch duringchromatographic assay. The application of near infra-red reflec-tance (NIR) or transmission (NIT) spectroscopy for rapid estimatesof non-starch polysaccharides are in vogue (Blakeney & Flinn,2005). Near infra-red reflectance spectroscopy provides fast, safe,and inexpensive analysis. It is, however, a comparative techniquethat relies on multivariate calibration of sample spectra and accu-rate reference analysis (Neas, Isaksson, Fearn, & Davies, 2002;Williams & Norris, 2001). It has the potential to be exploited as arapid analytical method for nutritionally important components,including polysaccharides.

6. The anti-nutritive effect of non-starch polysaccharides

The enzymes for NSP digestion such as b-glucanases and b-xylan-ases are scarce or not present in fish (Kuz’mina, 1996). Consequently,the dietary NSPs remain undigested and therefore negatively affectanimal performance. The adverse effect is associated with the vis-cous nature of NSPs, their physiological and morphological effectson digestive tract, interaction with epithelium, mucus and micro-flora of gut (Table 3). Subsequently, it has been reported that solubleNSPs, such as mixed-linked b-glucans present predominantly in oatsand barley, increase intestinal transit time, delay gastric emptyingand glucose absorption, increase pancreatic secretion, and slowabsorption. However, the insoluble NSPs, like pentosans (arabinoxy-lans and xylans), beneficially decrease transit time, enhance water-

holding capacity and assist in faecal bulking in non-ruminantanimals (Pluske, Kim, McDonald, Pethick, & Hampson, 2001). Thefactors associated with the detrimental effects of NSPs are discussedbelow.

6.1. Modulation in digesta viscosity

The solubility and molecular weight of NSPs determine the vis-cosity. The solubility is not specific to the sugar composition orlinkage present in NSPs but depends on the chemical structureand association of NSPs with the cell wall components. However,the physical effect of viscosity on digestion and absorption of nutri-ents appear to be similar regardless of the sources of NSPs. More-over, the binding of NSPs with the intestinal brush border increasesthe thickness of the unstirred water layer adjacent to the mucosa,leading to impaired nutrient digestion and absorption (De Lange,2000). Furthermore, increased endogenous intestinal secretion ofwater, nutrients and other electrolytes has been suggested as acause of reduced nutrient digestion (Choct, 1997). High viscosityalso increases residence time of the digesta and therefore increasesintestinal volatile fatty acid (VFA) production. The resulting drasticchanges in the gut ecosystem decrease nutrient digestion andeventually reduce performance (Choct et al., 1996). In rainbowtrout (Oncorhynchus mykiss), reduced nutrient digestibility oninclusion of dietary soluble NSP (guar gum) was associated withan increase in digesta viscosity (Storebakken, 1985). Similarly,inclusion of soybean NSPs in the diet of Atlantic salmon resultedin a relatively high viscosity in the intestinal content that was re-flected in the reduction of amino acid and lipid digestion (Refstieet al., 1999). Cumulative apparent absorption of amino acids, nitro-gen, and sulphur was slowed down in Atlantic cod (Gadus morhua)by dietary bioprocessed soybean meal, which was possibly due tothe high water-binding capacity of this soy product (Refstie et al.,2006).

Feeding tilapia (Oreochromis niloticus) with cereal grain NSPsled to augmentation in digesta viscosity and reduction in digestadry matter. Moreover, the absorption of minerals, especially so-dium ion, was significantly negatively correlated with digestaviscosity (Almirall, Francesch, Perez Vendrell, Brufau, & Esteve-Garcıa, 1995; Leenhouwers, Ortega, Verreth, & Schrama, 2007b).Similarly, inclusion of soluble NSPs in the diet of African catfish(Clarias gariepinus) induced large increases in digesta viscosityand thereby intestinal fermentation activity, digesta dry mattercontent and digestive organ weights and the nutrient digestibil-ity was adversely affected beyond a certain viscosity threshold(Leenhouwers, Adjei, Verreth, & Schrama, 2006; Leenhouwers,Ter, Verreth, & Schrama, 2007a). In addition, supplementationof galactomannan-rich endosperm of sesbania (Sesbania aculeate)seeds in the diet of common carp (Cyprinus carpio) resulted in in-creased viscosity in the intestinal content, thereby affecting thenutrient absorption and utilisation (Hossain, Focken, & Becker,2001). In general, the soluble NSPs are generally viscous in nat-ure, and therefore they enhance the viscosity of the diet as wellas intestinal digesta. The magnitude of viscosity development ingut with response to dietary NSPs varies among animals and alsodepends on the source of NSP (Montagne, Pluske, & Hampson,2003). Digesta viscosity in the proximal intestine of African cat-fish and Nile tilapia was considerably higher in groups fed ryethan in those fed maize and wheat (Leenhouwers et al., 2007a,2007b).

The age of fish may be an important factor determiningtheir sensitivity towards viscosity. The probable reason for thisage-related reaction is that a more developed intestinal micro-biota in older animals is able to utilise NSPs more efficiently(Choct & Kocher, 2000; Refstie et al., 2006a) than in youngerones.

A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1417

6.2. Alteration in gastric emptying and rate of passage

Dietary soluble NSPs increase the viscosity of digesta in mono-gastric animals and decrease the rate of passage, whereas theinsoluble NSPs, such as cellulose and hemicellulose, increase thepassage rate (Johansen, Knudsen, Sandstrom, & Skjoth, 1996).

Dietary inclusions of soluble NSPs reduce the rate of gastricemptying in fish, which can delay the intestinal absorption ofglucose (Bach Knudsen, 2001) and possibly of other nutrients. InAfrican catfish, inclusion of viscous cereal grains in the diet re-duced the plasma cholesterol and glucose levels (Leenhouwerset al., 2007a). In common carp fed diets containing sesbania endo-sperm, both muscle and plasma cholesterol levels were signifi-cantly lowered (Hossain et al., 2001). Also significant reductionin total cholesterol levels in blood plasma of rainbow trout, yellow-tail and Atlantic salmon fed diets containing NSP-rich soybeanmeals has been reported by various authors (Kaushik et al., 1995;Refstie et al., 1999; Shimeno, Hosokawa, YÅmane, Masumoto, &Uneno, 1992). A reduced blood cholesterol level is probably associ-ated with binding or trapping of bile salts in the gut, due to highviscosity, as also observed in rats fed with galactomannans fromguar gum (Moundras, Behr, Remesy, & Demigne, 1997).

6.3. Alteration of gut physiology, gut morphology, native gutmicroflora and mucus layer of gut

Apart from increasing the gut viscosity, the soluble NSPs elicitan anti-nutritive effect by modifying the gut functions. These ham-per the endogenous secretion of water, proteins, electrolytes andlipids (Angkanaporn, Choct, Bryden, Annison, & Annison, 1994).Non-starch polysaccharides can enhance bile acid secretion andsubsequently result in significant loss of these acids in the faeces(Ikegami et al., 1990). This can result in increased hepatic synthesisof bile acids from cholesterol to re-establish the homeostasis,which may ultimately influence the absorption of lipids and cho-lesterol in the intestine, resulting in lower blood cholesterol levels(Hossain et al., 2001). These impacts could lead to remarkablechanges in the dynamics of the gut physiology, ensuring poornutrient assimilation efficiency by the animal. Non-starch polysac-charides may also influence lipid metabolism in the intestine,through binding with bile salts, lipids and cholesterol (Vahouny,Tombes, Cassidy, Krichevsky, & Gallo, 1981). Hitherto, there areno reports on the direct inhibition of intestinal enzyme synthesisby NSPs but the activities of most enzymes may be reducedthrough coupling to NSPs or physical restriction of enzyme accessto substrates (Pettersson & Åman, 1989). Digestive enzyme activityof broiler chicks responded to diets supplemented with commer-cial NSPs (Iji, Saki, & Tivey, 2001). The jejunal maltase and sucraseactivities were highest in chicks that were fed an alginic acid-sup-plemented diet (low viscosity) and lowest in chicks fed the gumxanthan-supplemented diet (high viscosity). The activity of amino-peptidase N in the ileum was also stimulated by a highly viscousNSP diet. However, the uptake of amino acid L-tryptophan intobrush-border membrane vesicles was unaffected by NSP supple-ment. To our knowledge, no information is available on the directeffects of NSPs on fish gut physiology.

A number of authors have reported that dietary NSPs have a con-siderable impact on the gut anatomy and gut development. A pro-longed consumption of soluble NSPs is associated with increasedsize and length of the digestive organs in pigs (McDonald, 2001),chickens (Iji et al., 2001) and fish (Leenhouwers et al., 2006) accom-panied by a decrease in nutrient digestion. Pigs fed with high dietaryfibre food for 14 days resulted in significant reduction in concentra-tions of DNA in jejunum, ileum, and liver, indicating programmedcell death, while concentrations of RNA in the colon were signifi-

cantly greater (Jin, Reynolds, Redmer, Caton, & Screnshaw, 1994).These authors also reported enlargement in the width of intestinalvilli and increased depth of intestinal crypts in jejunum and ileum,and increased rate of cell proliferation and increased crypt depthin the large intestine. Since the crypts are the principal sites of cellproliferation in the intestinal mucosa (Baserga, 1985), an increasein the rate of crypt-cell proliferation, along with the reduction inconcentrations of DNA, may increase the rate of turnover of intesti-nal mucosal cells. It could be an explanation for the reduction in vil-lus height and crypt depth ratio observed in the small intestine ofpigs subjected to feed supplemented with high fibre (Jin et al.,1994). Similarly, administration of an oral dose of cellulose to pre-weaning pigs reduced villus length by approximately 15% in the jeju-num and ileum (Jin, 1992). Furthermore, McDonald (2001) reportedthat addition of sodium carboxymethylcellulose (CMC) to a highlydigestible cooked rice-based weaner diet (40 g/kg diet dry matter)for 13 days significantly increased the intestinal viscosity of digestawithin the small and large intestine, which led to decreased villuslength and increased crypt depth. Similar results have been reportedby Hopwood, Pethick, and Hampson (2002).

The shortening of villi results in an impaired absorption becauseshortening results in an absolute loss of intestinal surface area andcells. Since the osmotic water absorption is governed by nutrientabsorption, the decline in nutrient absorption caused by reductionof villi length eventually impairs the water absorption. Moreover,an increased crypt depth is associated with an increased watersecretion into the intestinal lumen. This large fluid amount in theintestinal lumen is generally not absorbed by the partially devel-oped large intestine of young animals, which may give rise to clin-ical symptoms of dehydration (Nabuurs, 1998). In chicken, feedingwith gum xanthan-supplemented diet for 14 days resulted indeepening of the jejunal crypts and reduction in villi height andvilli surface. Additionally, the viscous NSPs increased the weightof the small intestine. However, changes in the weight of visceralorgans were generally due to variation in rate of cell proliferation,cell size or protein synthesis (Iji et al., 2001). This was further con-firmed in this study by an increase in the protein:DNA ratio (cellsize) of the jejunum and by a decline in the DNA content (cell pop-ulation) of chickens raised on NSP-containing diets.

Conversely, Moore, Kornegay, Grayson, and Lindemann (1988)found that villus shape or surface morphology in jejunum of grow-ing pigs was not affected by a high-fibre diet, although some loss ofepithelial cells at the apex of the villi was observed. In rats, inges-tion of pectin (25 g pectin/kg of elemental diet for 14 days) signif-icantly increased villus height and crypt depth (Andoh, Bamba, &Sasaki, 1999). Supplementation of low viscosity CMC (40 g CMC/kg air-dried diet) to a cooked rice-based diet increased the smallintestinal villus and crypt depth in newly weaned pigs withoutaltering the shape of villi (McDonald, 2001).The above studieshighlight the effects of NSPs on gut morphology of monogastricanimals. It is also evident that information on fish is lacking.

Mechanisms for modifying gut morphology: The consumption ofan NSP-rich diet alters the intestinal physiology and anatomy byits ability to increase digesta viscosities. The rates of crypt-cell pro-liferation, cell migration along the crypt–villus axis, and cell extru-sion from the villous apex via apoptosis and cell sloughing controlthe dynamic process of small intestinal cell turnover. The presenceof high digesta viscosity in the lumen may increase the rate of vil-lus cell losses, leading to villus atrophy, a phenomenon associatedwith an increased crypt-cell production, and generally with in-creased crypt depth (Montagne et al., 2003).

The delay of digesta passage in the intestinal tract, as a result ofincrease in viscosity, may stimulate microbial fermentation in theintestine. The fermentation of NSP produces volatile fatty acids(VFA) as an end product. Administration of NSPs in the diet of tila-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-ers et al., 2007a, 2007b). Acetic acid is the prominent VFA producedin African catfish, which is in agreement with other studies on tila-pia and marine herbivores (Amirkolaie et al., 2006; Clements &Choat, 1995; Kihara & Sakata, 1997). Besides acetic acid, propionicand butyric acids are also produced following microbial fermenta-tion in fish but the concentration of these two acids varies amongfish species. A low concentration of propionic and butyric acid intilapia was observed (Amirkolaie et al., 2006) while a significanthigh amount of these acids was reported from marine herbivorousfish (Clements & Choat, 1995). The production of these organicacids in the intestinal tract may lower its pH, which in the longterm may disturb the normal microbiota prevailing in the gut. Be-sides, gastric acidity influences the bioavailability of dietary miner-als (Wood & Serfaty-Lacrosniere, 1992) by regulating chelation andcomplex formation and by altering the transport mechanisms ofminerals (Ravindran & Kornegay, 1993). Moreover, increase inthe residence time of digesta in the intestine following intake ofsoluble NSPs may decrease oxygen tension and favour the develop-ment of anaerobic microbiota (Choct, 1997). Although it is not fullyknown whether a sudden change of gut ecology is detrimental tothe efficiency of nutrient utilisation, the maintenance of health sta-tus relies mainly on normal endogenous microbiota. The normalmicrobiota confers many benefits to the intestinal physiology ofthe host. Some of these benefits include the metabolism of nutri-ents and organic substrate and the contribution to the phenome-non of colonisation resistance. However, when the delicatebalance of normal microbiota is upset, pathogens that arrive orthat have already been present but in numbers too small to causedisease take the opportunity to multiply. Furthermore, the prolifer-ation of some anaerobic organisms can lead to production of toxinsand deconjugation of bile salts which are essential for the digestionof fat (Carre, Gomez, & Chagneau, 1995).

Mucus is the protective layer of the entire gastro-intestinaltract, which is exposed to all chemical and physical forces of diges-tion. Several studies have correlated the dietary NSPs intake andmucin concentration in the gastro-intestinal tract. In pigs, inclu-sion of pea fibre in a wheat diet tended to increase the output ofmucins in the ileal digesta from 6.1 to 7.3 g per day for diets sup-plemented with 0 and 240 g of pea fibre per day, respectively (Lien,Sauer, & He, 2001). Similarly ileal glucosamine and galactosamineexcretion increased continuously with fibre intake (Reverter,Lundh, & Lindberg, 1999). When wheat bran (150 g/kg diet drymatter) was added to a protein-free diet for pigs, the ileal outputof galactosamine at the terminal ileum increased from 1.93 to4.13 g per day (Fuller & Cadenhead, 1991). Feeding CMC as a diges-ta viscosity-inducing non-fermentable polysaccharide to weanedpiglets for 15 days significantly increased viscosity of ileal digestaand ileal mucin concentration (Piel, Montagne, Sève, & Jean-Paul,2004). The mechanisms by which dietary NSPs modify mucin char-acteristics are not well understood. The physical scraping and pro-teolytic breakdown of mucus gels are the main factors releasingmucins into the gut lumen (Allen, 1981). Therefore, it could behypothesised that the erosion of mucus layer may be due to an in-crease in the bulk of digesta, stretching the intestinal mucosa andscraping mucin from the mucosa as they pass through the digestivetract.

It could be contemplated that effects reported above may alsoappear in fish but to date no such studies have been conductedin fish.

7. Effect of non-starch polysaccharides on nutrient metabolism

Feeding of NSPs negatively influences the metabolism and util-isation of dietary nutrients, like glucose, lipid, amino acid and min-

erals. This is because of a reduction in the rate of gastric emptying,leading to a depression in nutrient absorption (Bach Knudsen,2001). Moreover, Leenhouwers et al. (2006) explained that reduceddigestibility of nutrients is related to partial distribution of diges-tive enzymes in a viscous solution and a lowered flow at the muco-sal layer. Enhanced endogenous losses of nutrients and increase inthe thickness of the unstirred water layer adjacent to the mucosaalso lead to the diminution in nutrient digestion and absorption(De Lange, 2000).

7.1. Effect on glucose

The inclusion of NSPs in the basal diet of monogastric animalsincluding fish has been reported to delay the intestinal absorptionof glucose. In African catfish it was demonstrated that feeding dietscontaining rye at a level of 400 g/kg diet (dry matter) decreasedplasma glucose level (Leenhouwers et al., 2007a). In salmonid fish,inclusion of guar galactomannans and alginates as NSP sources inthe diet reduced the availability of glucose when compared toNSP-free diets (Storebakken, 1985; Storebakken & Austreng,1987). Significantly lower intestinal maltase activity in Atlanticsalmon has been reported on feeding defatted soybean meal con-taining NSPs at a level of 100 g/kg diet (dry matter) (Kraugerudet al., 2007). In fish, knowledge on the glucose digestibility inresponse to dietary NSPs is limited and only few studies have beenconducted to elucidate the effect of NSP on carbohydrate metabo-lism and absoption. However, a considerable number of reports areavailable in pigs supporting a negative effect of dietary NSP on glu-cose level. In growing swine the use of guar gum in basal feedhalved the rate of absorption of glucose in the jejunum (Rainbird,Low, & Zebrowskat, 1984). A reduction of 25% in the plasma glu-cose concentration in pigs fed with semipurified diets supple-mented with 40 g/kg guar gum in the diet (dry matter) has alsobeen reported (Sambrook & Rainbird, 1985). Furthermore, Nunesand Malmlof (1992) demonstrated that feeding 60 g/kg guar gumin the diet to swine reduced glucose absorption by 32%. Further-more, the inclusion of 60 g/kg guar gum in semipurified diet ofswine reduced the postprandial production of insulin by 30%, insu-lin-like growth factor-1 (IGF-1) by 58%, gastric inhibitory polypep-tide by 55% and glucagon by 41% (Nunes & Malmlof, 1992). Lowerblood glucose level was observed when trout were fed an experi-mental diet prepared by replacing 62.5% of fish meal protein withdetoxified Jatropha curcas kernel meal (DJKM) which contained16% NSP (Kumar, Makkar, & Becker, 2010).

7.2. Effect on protein

Inclusion of NSPs in the diet of fish has been well documented toreduce amino acid digestibility. The decrease in nitrogen utilisationefficiency following an NSP-rich diet is probably due to increase ofN secretion either endogenously and/or through intestinal bacteria.Leenhouwers et al. (2006) reported that inclusion of soluble NSPsfrom guar gum at a level of 40 and 80 g/kg in the diet (dry matter)of African catfish diet significantly increased digesta viscosity in theproximal and distal intestine. This increase in viscosity was accompa-nied by a reduction in the apparent digestibility coefficient of protein.Storebakken (1985) reported reduced protein digestibilities in rain-bow trout fed with 25–100 g guar gum per kg diet. African catfishreared with a high-viscosity rye-supplemented diet lowered the pro-tein digestibility much more than those fed with a low-viscositywheat-based diet (Leenhouwers et al., 2007a). However, it was in con-trast with the findings of Leenhouwers et al. (2007b) with tilapiawhere reduction in protein digestibility was observed more in fishfed with low-viscosity wheat than high-viscosity rye-based diet. Thissuggests that viscosity is not the only factor that explains the differ-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 itsmore herbivorous feeding habit (Leenhouwers et al., 2007b). Simi-larly, feeding salmon on soya diets with high levels of soluble NSPs re-sulted in reduced protein digestibilities (Refstie et al., 1999).Furthermore, sesbania endosperm, a leguminous seed at P7.2% indiet of common carp (Hossain et al., 2001) and at P5.8% in diet of tila-pia (Hossain, Focken, & Becker, 2003) substantially reduced the pro-tein efficiency ratio and protein productive value. Other studies thathave included purified soluble NSPs in fish diets also found reducedprotein digestibilities (Shiau, Yu, Hwa, Chen, & Hsu, 1988).

7.3. Effect on lipid and cholesterol level

Increasing the NSP content in the diet of monogastric animalshas been reported to decrease the utilisation of lipid. Increase indigesta viscosity caused by intake of an NSP-containing diet hasbeen shown to affect emulsification negatively, and to reduce lipol-ysis (Pasquier et al., 1996). Non-starch polysaccharides may entrapbile salts, thus reducing their efficiency in solubilising fats and con-sequently impairing lipid absorption (Ebiharam & Schneeman,1989). In African catfish, the digestibility of fatty acids was reducedconsiderably, when fed with wheat or rye at a level of 400 g/kg ofbasal feed (dry matter) (Leenhouwers et al., 2007a). Feeding ofcommon carp with sesbania endosperm (containing about 75% ofgalactomannan) at levels of P7.2% significantly reduced both mus-cle and plasma cholesterol levels (Hossain et al., 2001). Similar ef-fects were observed when tilapia was fed sesbania endosperm atlevels of P5.8% (Hossain et al., 2003). Moreover, reduced total cho-lesterol levels in blood plasma of Atlantic salmon fed diets contain-ing soybean meal have also been reported (Refstie et al., 1999),which could possibly be ascribed to NSPs present in soybean meal.Similarly, 62% of FM protein replacement by DJKM (detoxifiedjatropha kernel meal) resulted in lower lipid digestibility in trout,which could possibly be due to the presence of NSPs in DJKM(Kumar et al., 2010). The presence of NSPs in feeds has beenhypothesised to reduce fat absorption in trout by disturbing mi-celle formation in the gastro-intestinal tract (Øverland et al., 2009).

The hypocholesterolaemic response on NSP intake has also beenreported in Atlantic salmon (Salmo solar) fed with soybean mealcontaining NSPs at a level of 100 g/kg diet (dry matter) (Kraugerudet al., 2007). This is probably associated with binding of cholesterolwith bile salts in the gut. Moreover Potter (1995) described an-other possible mechanism for hypocholesterolaemia, as anenhancement of bile acid excretion and consequently creation ofan environment in which cholesterol is being ‘pulled’ from thebody. In this state, hepatic cholesterol metabolism alters to providecholesterol for enhanced bile acid synthesis.

7.4. Effect on minerals

Various components of NSPs interact with minerals and havebeen shown to decrease mineral absorption. Components of poly-saccharides and lignin that interact with minerals include the car-boxyl group of uronic acid, carboxyl and hydroxyl groups ofphenolic compounds and the surface hydroxyl of cellulose (Torre,Rodriguez, & Sauracalixto, 1995). Moreover, NSP-induced digestaviscosity has been shown to hinder mineral absorption (Van derKlis, Kwakernaak, & De Wit, 1995). Absorption of Ca, Mg, Na andP were considerably lower when African catfish were fed with adiet supplemented with rye (Leenhouwers et al., 2007a), whileonly Na absorption was significantly reduced when the same dietwas fed to Nile tilapia (Oreochromis niloticus) (Leenhouwers et al.,2007b). Elevated faecal sodium excretion in Atlantic salmon asan effect of feeding soy products was pointed out by Storebakkenet al. (1998). Likewise, in the same fish species a negative impactof defatted soyabean meal on K, Na, Zn and of native soy-NSP on

Cu, Fe and K utilisation were observed (Kraugerud et al., 2007).Furthermore, in rainbow trout Na excretion has been found toincrease proportionally with dietary cellulose level (Øvrum &Storebakken, 2007). In contrast to the above findings, no correla-tion was observed between NSP content in diet and sodiumexcretion in Atlantic salmon (Aslaksen et al., 2007).

Although dietary NSPs have mainly been reported to have neg-ative effects on mineral utilisation, epidemiological data suggeststhat intake of plant ingredients rich in NSPs can be a protective fac-tor against the abnormalities caused by metal toxicity. Despite anumber of studies conducted to elucidate the effects of dietaryNSPs on mineral bioavailability, little attention has been devotedto the understanding of their chelating mechanism.

8. Effect on growth performance and body composition

Low-protein soy products have been shown to induce negativeeffects on the digestibility of nutrients in salmon, which was prob-ably an effect of the viscosity caused by the NSP in the soybeanproduct (Refstie et al., 1999). Besides, in rainbow trout, tilapiaand Atlantic salmon, an increased water content of the gut inducedby the water-binding properties of dietary soluble NSP has beensuggested as the reason for the reduced nutrient digestibilitiesand diminished growth performance (Refstie et al., 1999; Shiauet al., 1988; Storebakken, 1985). Feeding rainbow trout a diet con-taining 10% guar gum, which contains high proportions of galacto-mannan, resulted in significant reduction of growth and dry matterand fat content in fish tissues. Moreover, in common carp and troutthe replacement of 75% and 62.5% of FM protein by DJKM respec-tively decreased growth performance. This adverse effect wasattributed to the presence of NSPs in DJKM (Kumar, Makkar, &Becker, 2008; Kumar et al., 2010). Dietary inclusion of sesbaniaendosperm at levels of 7.2%, 10.8% and 14.4% in the feed of com-mon carp resulted in reduced body weight gains that were 57%,48% and 39%, respectively of the control diet (Hossain et al.,2001). Additionally, the inclusion of sesbania endosperm influ-enced whole body proximate composition of fish, with signifi-cantly increased whole body moisture, reduced lipid and grossenergy content (Hossain et al., 2001). Moreover, similar observa-tions of higher whole body moisture and lower lipid content incommon carp fed diets containing various levels of rape seed, mus-tard oil-cake, linseed and sesame meal have been made (Dabrow-ski & Kozlowska, 1981; Hossain & Jauncey, 1989). The study ofHossain et al. (2001) showed no significant difference in thehepatosomatic index of fish. It is in agreement with other studieson fish and poultry in which the effects of feeding soluble NSPson relative liver weight were insignificant (Iji et al., 2001; Leen-houwers et al., 2006; Storebakken, 1985). Conversely, the studyconducted on African catfish showed stimulating effects of guargum on stomach and intestine weights (Leenhouwers et al.,2006). A feeding trial on tilapia showed that weight gains of fishfed diets containing 5.8%, 8.7% and 11.8% sesbania endospermwere 82%, 73% and 64%, respectively compared to the control diet(Hossain et al., 2003) and these fish also had significantly higherwhole body moisture, lower lipid and lower gross energy contents.It was also noticed that the reduction in whole-body lipid wasmore pronounced in the diets containing higher levels of endo-sperm. It may be because of the thickening of the fluid layer closestto the mucosal wall, thereby preventing contact of digestiveenzymes with the substrates and the formation of the micelles re-quired for the lipid absorption (Wang, Newman, Newman, & Hofer,1992). In contrast to the above findings, Leenhouwers et al. (2007a)reported no effect on the growth performance in African catfishwith increase in intestinal viscosity. A previous study with Africancatfish 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(Leenhouwers et al., 2006). These differences could be attributed tofactors such as differences in NSP concentration, NSP structure,concentration of other dietary components, fish species, and ageof fish (Leenhouwers et al., 2007a; Petersen, Wiseman, & Bedford,1999).

9. Non-starch polysaccharides and effect on gelatinisation

Fish in general have a limited capacity for carbohydrate utilisa-tion and processing methods, such as gelatinisation, have beenreported to improve the nutrient bioavailability to the fish. Gela-tinisation is a thermal modification of raw dietary carbohydrates.During this process carbohydrate granules are modified in such away that their susceptibility to enzymatic action increases (Kumar,Sahu, Pal, Choudhury, & Mukherjee, 2006), making digestion morecomplete. Carbohydrate gelatinisation and its impact on digestibil-ity and growth have been well documented in fish such as carp(Kumar et al., 2006) and rainbow trout (Podoskina, Podoskin, &Bekina, 1997). Wheat, being the major source of starch, also con-tains a low amount of NSPs. Arabinoxylan, the main NSP in wheatflour, is reported to hinder the gelatinisation and retrogradationproperties of wheat starch, due to its water-binding capacity andhigh viscosity (Shogren , Hashimoto, & Pomeranz, 1987). Moreover,addition of an isolated NSP-rich fraction to starch markedly affectsstarch gelatinisation and pasting properties (Sasaki, Yasui, &Matsuki, 2000). Furthermore, Tester and Sommerville (2003)reported that soluble NSPs restrict the gelatinisation of maizestarch and wheat starch by reducing hydration of the amorphousregions and consequently limiting depression of the glass transi-tion temperature within these regions. This suggests that the effectof NSPs on the gelatinisation event, in general is mediated viareducing the volume fraction of water in the system and restrictingwater availability and mobility due to the hydration process(Tester & Sommerville, 2003). The plant tissues containing starchare expected to contain NSPs, which could hinder the effect ofprocessing on starch digestibility. Better understanding of theeffects of different NSP types on gelatinisation would aid in theefficient utilisation of plant carbohydrates in fish nutrition.

10. Interaction with antibiotics

The efficiency of NSP utilisation by fish is dependent on the nat-ure of the microbial population residing in the gut. It, could, there-fore, be speculated that the supplementation of antibiotics thatalter the gut microflora would have an influence on dietary fibreutilisation (Annison & Choct, 1991). A large body of evidence sug-

Table 4Role of b-glucan in health and production of fish and crustaceans (adapted from Kumar e

Fish/crustacean Route of administration Advantage

Fish Feed or injection � Enhances prod� Acts in synergy

Fish and shrimp Injection and oral � Acts as a true aHalibut larvae Immersion � Increases surviCoho salmon Injection or oral � Protects againsShrimp Feed � Increases growFish and shell fish Feed � Acts in synergyShrimp post larvae Immersion � Enhances the sPenaeus monodon Feed � Protects agains

� Increases surviMacrobrachium rosenbergii larvae Bath treatment (10 mg/L) � Increases lysos

� Protects againsCarp Injection (500 mg/kg) � Enhances proteAsian catfish Feed � Enhances imm

gests that the anti-nutritive activity of NSPs in poultry is related tothe gut microflora, as addition of antibiotics to diets increasednutritive value of diets (Langhout & Schutte, 1995). Moreover, inswine, supplementation of virginiamycin at a level of 11 ppm indiets containing 50% oats improved dry matter, energy, hemicellu-lose and cellulose digestibilities. Furthermore, the antibiotic sup-plementation also improved absorption and retention ofminerals, such as Ca, P, Mg, Cu, Fe, Zn and Mn (Ravindran & Korne-gay, 1993). Similarly, 0.7% nebacitin in a pig diet containing 10.5%fibre enhanced the apparent N digestibility by 12%; salinomycin ata level of 82 mg/kg of a diet (dry matter) containing 20% wheatbran increased P absorption (Moore, Kornegay, & Lindemann,1986). Only a very limited number of works has been done onthe interactions of NSPs and antibiotics in fish. In Atlantic salmon,soybean meal-induced enteritis was not ameliorated by adding thebroad-spectrum antibiotic oxytetracycline to the diet at 3 g/kg (drymatter) (Bakke-McKellep et al., 2007). In the distal intestinal mu-cosa of fish fed both soybean meal and soybean meal supple-mented with oxytetracycline, a significant increase in theproliferative compartment length as well as apparent increases inthe number of cells undergoing cellular repair processes and apop-tosis were observed as pathophysiological responses related toenteritis induced by the soybean meal (Bakke-McKellep et al.,2007). In the same species, it was seen that soybean meal supple-mented with oxytetracycline resulted in reduced relative liverweight and did not modify the responses to dietary soybean mealinfluencing gut morphology, hydrolysis of protein and starch, andabsorption of amino acids, nitrogen and sulphur (Refstie et al.,2006b).

11. Purified non-starch polysaccharides as immunostimulants

Immunostimulants are chemical substances that activate thegeneralised immune response system of animals. Such substancesare also known to render animals more resistant to infectious dis-eases and reduce the risk of disease outbreaks if administered priorto stress-causing situations. NSP, such as b-1,3-glucans, acts as animmunostimulant and thus improves health, growth and generalperformance of many different animal groups, including farmedshrimp, fish and terrestrial animals (Kumar et al., 2005). Supple-mentation of b-1,3 glucan in diets enhances non-specific cellulardefence mechanisms by increasing the number of phagocytesand the bacterial killing activity of macrophages in rainbow trout,Atlantic salmon, catfish, and carp, and also by the macrophagemediated superoxide anions production (Kumar et al., 2005). In amajor Indian carp, Labeo rohita, yeast glucan (b-1-3 glucan) hasbeen observed to enhance the phagocytic activity of leucocytes

t al., 2005).

uction of antibodies against pathogenswith antibioticsEnhances resistance to bacterial disease and efficacy of vaccine

djuvant and enhances antibody productionval ratet Aeromonas salmonicidath, reduces mortality, and increases feed utilisation

with vitamin-Curvival ratet white spot syndrome virus infectionval rateomal activityt Vibrio alginolyticusction against Aeromonas hydrophila

unity against Aeromonas hydrophila

Table 5Non-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� Hydrolysis of b-glucans� Decrease in intestinal viscosity� Better emulsification and flexibility of feed materials

� Improvement in nutritional quality of animal feed� Improvement in performance of ruminants and monogastrics

b-Glucanase and xylanase � Hydrolysis of cereal b-glucans and arabinoxylans� Decrease in intestinal viscosity� Release of nutrients from grains

� Improvement in feed digestion and absorption, and in weight gain ofbroiler chickens and hens

Hemicellulase with highxylanase activity

� Increase in nutritive quality of pig feeds � Reduction in the cost of pig feeds� Reduction in the cost of feeds for pigs

Cellulases, hemicellulases andpectinases

� Partial hydrolysis of plant cell wall during silage andfodder preservation

� Contribution to production and preservation of high quality fodder forruminants� Improvement in quality of grass silage

A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1421

and stimulates generation of reactive oxygen species (ROS) inphagocytes (Ali, Karunasagar, Pais, & Tauro, 1996). A number ofstudies have shown that b-1-3/1-6-glucans enhance the biologicalactivity of shrimp haemocytes and improve growth, survival rateand feed conversion efficiency under experimental conditions(Table 4). b-glucans have been successfully used to increase theresistance of shrimp Penaeus japonicus against vibriosis (Kumaret al., 2005). Penaeus monodon when fed with b-glucan at 0.2%(w/w of the feed) significantly increased phenol oxidase, the num-ber of haemocytes and the bacterial killing activity against vibrio-sis, white spot syndrome virus, Vibrio damsela and V. harveyi andalso enhanced survival and immunity during brood stock rearing(Chang, Su, Chen, & Liao, 2003). Macrobrachium rosenbergii post lar-val stage showed enhanced growth and resistance to V. alginolyti-cus as a result of dietary administration of b-glucan (Misra et al.,2004). However, the long term administration of b-glucan in fishand shellfish can reduce the immune response to basal levels and

Fig. 6. Modes of action of non-starch polysaccharide d

also reduce the effect on those bacteria that are resistant tophagocytosis.

Mannose units that comprise glucomannans, belong to the cat-egory of compounds that adhere to receptors used by pathogenicmicrobes as the first step of colonisation of the gut. Therefore, sup-plementation of mannose in basal feed of animals may contributeto better health by interfering with colonisation and growth ofpathogens in the gut (Raa, 2000).

Levan, a natural polymer of fructose with b (2,6) linkages pro-duced extracellularly by many microorganisms like Zymomonasmobilis, Bacillus subtilis, Bacillus polymyxa, and Acetobacter xylinum,is shown to have immunostimulating properties in fish (Guptaet al., 2008; Rairakhwada et al., 2007). Dietary levan supplementsat a concentration of 0.5% have been reported to activate non-spe-cific phagocytes in Cyprinus carpio juveniles which resulted inhigher survival rate when challenged with Aeromonas hydrophila(Rairakhwada et al., 2007). Likewise the histological study strongly

egrading 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-fects of infection by A. hydrophila in Labeo rohita juveniles.

12. Non-starch polysaccharides degrading enzymes

Monogastric animals including fish lack the intestinal enzymesfor the degradation of NSPs. The improvement in the digestibilityof NSPs is achieved by supplementation of NSP-degrading enzymesin the diet. Such an approach has successfully been used in poultrydiets but little work showing the potential of NSP-degrading en-zymes in fish is available (Ai et al., 2007).

Various kinds of NSP-degrading enzymes have been reported inanimal feed, which include cellulase, hemicellulase, xylanase, pec-tinase, b-glucanase and a-galactosidase. Their effects and benefitsare illustrated in Table 5. b-Glucanases and xylanases have beensuccessfully used in monogastric diets to hydrolyse NSPs, such asbarley b-glucans and arabinoxylans (Cowan, 1996). Moreover Yin,Baidoo, Schulze, and Simmins (2001) reported that inclusion ofb-glucanase (600 units/kg diet) and xylanase (745 units/kg diet)in pig feeds containing hull-less barley decreased the viscosity inthe distal part of the small intestine, lowered the plasma ureanitrogen concentration and increased the apparent ileal digestibil-ity of energy and some amino acids. A similar beneficial effect ofNSP-degrading enzymes in pigs has been reported by Yin et al.(2000). Besides, addition of NSP-degrading enzymes during feedproduction was found to degrade NSPs and markedly improvedthe digestion and absorption of feed components as well as growthperformance in broiler chickens (Choct, Hughes, Trimble, Angka-naporn, & Annison, 1995; Ghorbani, Fayazi, & Chaji, 2009). Almirallet al. (1995) showed that broiler chicks fed with high viscosity bar-ley had lower amylase and lipase activities in the digesta, whileaddition of b-glucanase in the diet increased activities of the twoenzymes and of trypsin as well. This observation was consistentwith the result of Li, Li, and Wu (2009) with tilapia (Oreochromisniloticus � Oreochromis aureus) where 1 g/kg of NSP-degradingenzyme (50 FBG (fungal b-glucanase units)/g; Roche Shanghai) in-creased activity of amylase in the hepatopancreas and intestine by11.4% and 49.5%, respectively. The application of Natugrain-blend�

(containing b-glucanase and b-xylanase) at concentrations of 75,150 and 300 ll/kg in diets containing 30% wheat or dehulled lupinhad no remarkable effect on dry matter, energy or protein digest-ibilities when fed to silver perch (Stone, Allan, & Anderson,2003a). According to Stone et al. (2003a) the missing effect of Nat-ugrain-blend� inclusion on nutrient digestibility could be due tothe intolerance of silver perch to high levels of galactose and xylosein their blood (Stone, Allan, & Anderson, 2003b). Prolonged eleva-tion of blood galactose has been reported to reduce growth perfor-mance in carp (Shikata, Iwanaga, & Shimego, 1994).

The pretreatment of dietary plant materials with exogenouscarbohydrases (a-amylase, b-glucanases and b-xylanases) en-hances the energy digestibility by releasing more glucose, galact-ose and xylose (Kumar et al., 2006). The supplementation of twoNSP-degrading enzymes, for example 400 mg ‘VP’ (contains mainlyglucanase, pentosanase and cellulase, each at 50 IU per g) and800 mg ‘WX’ (contains mainly xylanase, 1000 IU per g) in basalfeed of Japanese seabass (Lateolabrax japonicus) significantly en-hanced the specific growth rate, feed efficiency ratio, nitrogenretention and reduced ammonia excretion (Ai et al., 2007).

12.1. Mechanism responsible for enzymic degradation of non-starchpolysaccharides

The mechanisms by which exogenous NSP-degrading enzymesenhance nutrient digestion and utilisation from plant proteins infish have not yet fully understood. However, based on various re-

ports three types of mechanisms could be suggested for the actionof NSP-degrading enzymes (Fig. 6). These are discussed below.

12.1.1. Disruption of cell wall integrityThe cell wall in cereals and legumes is constructed mainly of

small amounts of cellulose, hemicellulose and arabinoxylan withminor b-glucan components. The activity of exogenous NSP-degrading enzymes creates ‘holes’ in the cell wall. This allowswater hydration and permits pancreatic proteases and amylasesto act, enabling better digestion of the starch and protein. Xylan-ases and to a lesser extent cellulases have been proven most effec-tive in broilers (Leslie, Moran, & Bedford, 2007). Mannanases andpectinases have shown good results in a soy-based diet (Jackson,Geronian, Knox, McNab, & McCartney, 2004). However, in a corn-soy-based diet xylanase and glucanase were more effective inbreaking down the insoluble fibre fraction. Moreover, Li, Sauer,and Huang (1996) suggested that the improvement in digestibilityof nutrients in barley-based diets with cellulase addition for youngpigs was due to the increased degradation of b-linked componentsin barley, which made them available to the host animal.

12.1.2. Reduction of digesta viscosityStudies on monogastric animals have shown that reduced

digesta viscosity due to NSP-degrading enzyme supplementationis the main factor responsible for the observed enhanced perfor-mance response on feeding plant materials rich in NSPs (Cowieson,Singh, & Adeola, 2006). However, Partridge (2001) demonstratedthat pig small intestinal viscosity is relevant for its performancebut it is of a lower order of importance than for broilers. To ourknowledge no study is available which illustrates the effect of sup-plementing NSP-degrading enzymes on the intestinal viscosity infish fed NSP-containing diets.

12.1.3. Stimulation of bacterial populationAddition of NSP-degrading enzymes in feed breaks down plant

cell wall carbohydrates and reduces chain length, producing smal-ler polymers and oligomers. These fragments further become smallenough to act as a substrate for bacterial fermentation that can bebeneficial with VFA production and altering the bacterial popula-tion. Several studies have shown that applications of exogenousenzymes significantly alter VFA production and the populationprofiles of gut-associated microflora (Bedford & Apajalahti, 2001).Yin et al. (2000) studied the effects of xylanase supplementationin a wheat-bran-based diet in young pigs, and found that the en-zyme addition increased the ileal production of VFA.

However, care must be taken while selecting the feed enzymebecause some products can be overdosed and reduce the size ofthe oligosaccharides down too far, to monosaccharides. If excessmonosaccharides are produced, it may result in osmotic diarrhoeaand/or poor performance (Schutte, 1990).

Currently, the use of various exogenous feed enzymes is consid-ered to reduce FM inclusion by around 5% in most aquafeeds andthere is a potential to reduce further the demand for FM by theaquaculture sector in the coming years (Felix & Selvaraj, 2004).However, lack of information on the nutritive value and the con-tent and nature of anti-nutrients in feed ingredients make difficultthe selection of suitable enzymes or enzyme mixtures as feed addi-tives. Further studies are required in this area. Another constraintis that feed enzymes are proteins, which are substrate specific,work within a particular pH range and are themselves potentiallyopen to digestion by endogenous enzymes. In the stomach pH islow and suited primarily to protein digestion (Yin et al., 2001).Consequently it is crucial that enzymes are protected to a sufficientextent against proteolysis and are available at the targeted site ofactivity (Yin et al., 2001).

A.K. Sinha et al. / Food Chemistry 127 (2011) 1409–1426 1423

13. Conclusions

In aquaculture the use of plant-based protein is increasing at afast pace due to its relatively low cost and ample availability. Con-sequently, the use of non-starch polysaccharides (NSPs), a class ofanti-nutrients present in plant-based diets, will increase in the fu-ture. Currently there is relatively little information on the effects ofdietary NSP on fish nutrition and physiology. Non-starch polysac-charides, with their high water-holding capacity can affect digestaviscosity in fish. High digesta viscosity, produced as a result ofNSPs, delays gastric emptying and feed transit time, and decreasesinteraction of the intestinal enzymes with feed macromolecules,resulting in decreased nutrient availability. Eventually this leadsto decreased growth performance. Non-starch polysaccharides alsochange the gut ecosystem, which could be beneficial or deleteriousfor the animals. The extent of fermentation of NSPs in fish intes-tine, the change in gut microbial ecology and the factors leadingto adverse or beneficial effects in fish are not clear.

The addition of NSP-degrading enzymes (NSPases) in diets con-taining plant sources could play a vital role in improving nutrientutilisation in fish, as observed for other monogastric animals. Theuse of such enzymes may offer feed producers a means to cut downon feed costs. Different plant sources contain different types ofNSPs. The enzymes responsible for degradation of NSPs would dif-fer, depending on the nature of the NSPs. The efficacy of the en-zymes would also differ with the physiochemical conditions ofthe intestine and in particularly with the pH, which could differfrom one fish species to another. In addition, the enzyme shouldalso be resistant to intestinal proteases and should be active atthe target site of action. Therefore, systematic studies are neededfor better exploitation of NSP-degrading enzymes. Future studiesshould also be directed towards using purified NSPs as immuno-stimulants in commercial fish rearing.

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