Role of non-starch polysaccharides (NSP) and indigestible oligosaccharides in fish nutrition

Plant-based ingredients are the most cost-efficient alternative feed for fish in the aquafeed industry. Non-starch polysaccharides (NSPs) reduce the digestibility and bioavailability of nutrients in fish. NSPs are indigestible carbohydrates that can promote microbial growth in the gut. These prebiotics have shown beneficial effect on fish growth and health. These changes in GIT altogether stimulate the immune system, thus, enhancing the host’s protection against infections.

Ranju Kumari
Mfsc scholar
Department of Fish Nutrition and Feed Technology, ICAR-Central Institute of Fisheries Education, Mumbai, India

Feed costs account for over 50% of the variable costs in most aquaculture operations, therefore applying the best feeding strategy can have a significant impact on optimizing profit, which is the primary goal of commercial aquaculture. Fish meal due high protein content and favourable amino acid profile is an important protein source among commercial feeds and is preferred by fish culturists, however, due its restricted production (which increases its cost), the use of plant-based ingredients is increasing currently. Plant-based ingredients so far are the most cost-efficient alternative feed for fish in the aquafeed industry. This has increased the dietary content of indigestible carbohydrates such as non-starch polysaccharides (NSP) or dietary fibers which reduces the feed digestibility and bioavailability of nutrients.

Non-starch polysaccharides (NSPs) are a complex group, composed predominantly of linked monomers of hexoses and pentoses, e.g., galactose, glucose, arabinose, xylose and mannose that cannot be digested in fish and hence reduces the apparent digestibility of the diet and has negative impacts on growth. This is because the enzymes such as β-glucanases or β-xylanases that digest NSPs are scarce or absent in fish. Thus, the dietary NSPs remain indigestible and cannot be used as an energy source. NSP-containing diets in fish has slower rate of gastro-intestinal passage and also shown to reduce the availability of nutrients.

Through proper management of the NSPs in these plant materials, plant resources can very well be utilised as cheaper fish feed ingredients. The utilisation of exogenous enzymes (β-glucanases and β-xylanases) in feed processing decrease the negative effects of NSP and thus improve the nutritive value of feed. Moreover, NSPs such as β-glucans and mannose promote colonisation and microbial growth in the gut, thereby acting as immunostimulants.

The hydrolysed products of NSP, so called as indigestible oligosaccharides (or prebiotics) have shown beneficial effect on fish growth and health. Indigestible oligosaccharides are small fragments of carbohydrates (in between of simple sugars and polysaccharides) or oligosaccharides of galactose, fructose or mannose that selectively stimulate the growth or activity of one or a limited number of bacterial species, already present in the gut and thus improve host health and act as “prebiotics” (promote the growth of beneficial microbes). A probiotic i.e., a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance can be directly given along with feed, however, the probiotics strains can remain dominant in the gastrointestinal tract only during the dietary treatment. Such an exogenous addition of a single probiotic will result in long-term colonisation of the gut, especially when the strains used do not belong to the normal dominant intestinal flora. In those cases, the stimulation of specific indigenous microflora by supplementing fish feed with indigestible carbohydrates that act as prebiotic is more fruitful.

Indigestible carbohydrates could either be given to fish indirectly as NSP supplemented with exogenous enzymes in the feeds or included in the feed as prebiotics.

NON-STARCH POLYSACCHARIDES (NSPs)
Non-starch polysaccharides are long polymeric carbohydrate chains containing up to several hundred thousand monomeric units, excluding α-glucans (starch). NSPs in aquaculture feeds are present as an integrated part of the cell wall (upto 90% NSPs) of plant ingredients and also in a purified soluble form, such as guar gum, to stabilise the pellet. The most abundant plant cell wall NSPs include cellulose, hemicellulose and pectins; while fructans (inulin), glucomannans and galactomannans are not so abundant as of those aforesaid and serves as the storage polysaccharides within the plants. Mucilages, alginates, exudate gums, β-glucans and various modified polysaccharides are other constituents of the NSPs.

The main difference between NSPs and starch is that, starch is composed entirely of glucose monomers, which are linked by α-glycosidic bonds while NSPs are composed of different kinds of monomers, which are linked predominantly by β-glycosidic bond. The difference in bonding structure has profound effects on digestibility, as different classes of enzymes are required for the hydrolysis of α- and β-glycosidic bond. The predominant starch digestive enzymes are α-amylase, α-glucosidase and oligo-1-6-glucosidase. In combination, these enzymes specifically hydrolyse the α-glycoside bonds of starch to yield glucose. On the other hand, the enzymes required to digest NSP, such as β-glucanase and β-xylanases, are very scarce or even absent among fish species. The physiological impact of individual NSPs is dependent on the sugar residues present and nature of the linkage present between these residues.

Classification of NSPs
NSPs are classified into three main groups:
• Cellulose,
• Non-cellulosic polymers and
• Pectic polysaccharides.
Non-cellulosic polymers include arabinoxylans, mixed-linked β-glucans, mannans, and xyloglucan while pectic polysaccharides include polygalacturonic acids substituted with arabinan, galactan and arabinogalactan (Table 1).

NSPs can be broadly classified into:
• Insoluble fibers include cellulose, hemicellulose and pentosans like xylans and arabinoxylans, and
• Soluble fibers include mixed linked b-glucans, galactomannan (guar-gum) and pectins.

Role of NSPs in fish nutrition
The adverse effect of NSP is associated mainly with solubility of NSPs which depends on their chemical nature and their association with the cell wall components and which in turn is responsible for their viscous nature (due to which they do not move easily). Soluble NSPs increases the viscosity, which increases intestinal transit time, delay gastric emptying and glucose absorption, increase pancreatic secretion, and slow absorption.

Insoluble fibers are the NSP constituents of the cell wall that shield the substances inside the cell from the effect of the digestive enzymes and thus reduce their digestibility. However, insoluble fibers decrease transit time, enhance water holding capacity of the digesta (chyme), and add to the bulk of faeces in non-ruminant animals.

1. Effect on transit time, feed intake, nutrient digestion and absorption
Soluble NSPs bind with the intestinal brush border and increase the thickness of the unstirred water layer adjacent to the mucosa. This increases the viscosity of intestinal content which decreases the intensity of intestinal contractions, which in turn slows down the transit time of the digesta or chyme through the small intestine. Slow passage of digesta through the digestive tract, delays the gastric emptying time which results in reduced feed intake.

Due to reduced intestinal peristalsis a certain part of water contained in the chyme forms a hydration sphere around the intestinal content such that the digestive enzymes are not able to enter the intestinal lumen and also the mixing of enzymes with the intestinal content decreases. This decreases the efficiency of digestion.

As no digestive enzymes exist for the breakdown of cellulose and other NSPs, volatile fatty acids (VFA) like acetate, propionate and butyrate are produced in large amount in the gastrointestinal tract (GIT) by microbial fermentation of carbohydrates and endogenous substrates such as mucus. Such drastic change in gut ecosystem decreases nutrient digestion. Increased viscosity also causes shortening of intestinal villi and decreases their thickness which reduces the intestinal absorption surface area.

2. Effect on nutrient metabolism
NSP influence the metabolism and utilisation of dietary nutrients like glucose, lipid, amino acids and minerals. It also affected the distribution of digestive enzymes, gastric emptying rate, nutrients absorption and digestion.

• Effect on glucose
Diet containing high amount of soluble fiber increases viscosity of intestine content. This lowers the activity of digestive enzymes (like intestinal maltase) on substrate (say, maltose), which in turn reduces the availability of glucose and thus delays the intestinal absorption of glucose.

• Effect on lipid and cholesterol level
NSPs may bind or entrap bile salts (that cause emulsification of fats), thus reducing their efficiency of solubilizing fats. This disturbs micelle formation, thereby impairing lipid absorption in the GIT.

Increased viscosity in the intestine also increases bile acid excretion. As cholesterol is the precursor of bile acids so, it alters the hepatic cholesterol metabolism to provide cholesterol for enhanced bile acid synthesis and thus reduces the cholesterol content in the body (hypocholesterolaemia).

• Effect on protein and body growth
High fiber content in the diet increases abrasion of the intestinal cells and also increase the secretion of mucin which is compensated by the increase in excretion of endogenous protein or endogenous nitrogen particularly, threonine, this also decreases the utilization of nitrogen. This limits the synthesis of body proteins and thus lowers the efficiency of body growth.

• Effect on minerals
Components of polysaccharides that interact with minerals include the carboxyl group of uronic acid, carboxyl and hydroxyl groups of phenolic compounds and the surface hydroxyl of cellulose. Moreover, NSP-induced digesta viscosity has been shown to hinder mineral absorption mainly, sodium ions.

3. Effect on gelatinization
Fish in general have a limited capacity for carbohydrate utilisation and processing methods, such as gelatinisation, have been reported to improve the nutrient bioavailability to the fish. Gelatinization is a thermal modification process that modifies carbohydrate granules in such a way that their susceptibility to enzymatic action increases, making digestion more complete.

Wheat, being the major source of starch, also contains a low amount of NSPs. Arabinoxylan is the main NSP in wheat flour. NSPs due to its water-binding capacity (or hydration process) restricts water availability to the amorphous regions (wheat starch) and thus reduces the temperature within these regions and hinders gelatinization.

Figure 1. Effects of mixed prebiotics in aquaculture species. – Source: (Wee et al. 2022)

4. Purified non-starch polysaccharides as immunostimulants
Immunostimulants are chemical substances that activate the generalised immune response system of animals. NSP, such as β-1,3-glucans, acts as an immunostimulant. Supplementation of β-1,3 glucan in diets enhances non-specific cellular defence mechanisms by increasing the number of phagocytes and the bacterial killing activity of macrophages.

Mannose units that comprise glucomannans, belong to the category of compounds that adhere to receptors used by pathogenic microbes as the first step of colonisation of the gut. Therefore, supplementation of mannose in basal feed of animals may contribute to better health by interfering with colonisation and growth of pathogens in the gut.

INDIGESTIBLE OLIGOSACCHARIDES
Certain indigestible oligosaccharides are not digested in the upper part of the gastrointestinal tract, but are selectively fermented by bacteria like Bifidobacteria Lactobacilli and Bacteroides in the colon and promote the growth of these beneficial bacteria which in turn confer health benefit to host. Thus, these natural feed ingredients act as prebiotics. They include resistant inulin and oligofructose, transgalacto-oligosaccharides (TOS), mannan oligosaccharide (MOS) and lactulose.

Role of Indigestible oligosaccharides or prebiotics in fish nutrition
Prebiotic supplementation in fish feed may help in changing the community of bacteria in gastrointestinal tract, improving immune system and increasing growth rate.

1. Effects of prebiotics on immune system
Prebiotics can modify the GI tract microbial community to enhance non-specific immune responses. Indigestible oligosaccharides serve as substrate for the growth and proliferation of anaerobic bacteria, mainly the Bifidobacteria and are fermented by these bacteria in the caeco-colon region. During fermentation of prebiotics by beneficial bacteria (probiotics), short chain fatty acids (SCFA) are produced which cause acidification of the colonic content (or reduction of gut pH). Lower pH values inhibit the growth of certain pathogenic bacterial species while stimulating the growth of the Bifidobacteria and other lactic acid species.

Other changes include secretion of antimicrobial substances; blocking of adhesion sites; attenuation of virulence; blocking of toxin receptor sites; competition for nutrients, and suppression of toxin production. These changes in GIT altogether stimulate the immune system, thus, enhancing the host’s protection against infections.

2. Prebiotic may have the role of supplying energy for the host
Prebiotics are selectively fermented by probiotic bacteria e.g. Bifidobacteria, Lactobacillus and Bacteroides to produce short chain fatty acids (acetate, butyrate, propionate) and lactate. Short chain fatty acids are absorbed through the intestinal epithelium and act as an energy source for the host, whereas lactate enters the liver and is used as precursor for gluconeogenesis.

3. Effect on mineral absorption
Minerals such as calcium, magnesium and iron are not absorbed in the small intestine and so reach the colon, where upon fermentation of prebiotics, they are released from the carbohydrate matrix and are absorbed.

References
1. Sinha, A. K., Kumar, V., Makkar, H.P., De Boeck, G., & Becker, K. (2011). Non-starch polysaccharides and their role in fish nutrition – A review. Food Chemistry, 127(4), 1409-1426.
2. Yousefian, M., & Amiri, M.S. (2009). A review of the use of prebiotic in aquaculture for fish and shrimp. African Journal of Biotechnology, 8(25).
3. Wee, W., Hamid, N. K. A., Mat, K., Khalif, R. I. A. R., Rusli, N. D., Rahman, M. M., … & Wei, L. S. (2022). The effects of mixed prebiotics in aquaculture: A review. Aquaculture and Fisheries.