Amit Kumar Sinha

Dietary Roles of Non-Starch Polysachharides in Human Nutrition: A Review

Nonstarch polysaccharides (NSPs) occur naturally in many foods. The physiochemical and biological properties of these compounds correspond to dietary fiber. Nonstarch polysaccharides show various physiological effects in the small and large intestine and therefore have important health implications for humans. The remarkable properties of dietary NSPs are water dispersibility, viscosity effect, bulk, and fermentibility into short chain fatty acids (SCFAs). These features may lead to diminished risk of serious diet related diseases which are major problems in Western countries and are emerging in developing countries with greater affluence. These conditions include coronary heart disease, colo-rectal cancer, inflammatory bowel disease, breast cancer, tumor formation, mineral related abnormalities, and disordered laxation. Insoluble NSPs (cellulose and hemicellulose) are effective laxatives whereas soluble NSPs (especially mixed-link β-glucans) lower plasma cholesterol levels and help to normalize blood glucose and insulin levels, making these kinds of polysaccharides a part of dietary plans to treat cardiovascular diseases and Type 2 diabetes. Moreover, a major proportion of dietary NSPs escapes the small intestine nearly intact, and is fermented into SCFAs by commensal microflora present in the colon and cecum and promotes normal laxation. Short chain fatty acids have a number of health promoting effects and are particularly effective in promoting large bowel function. Certain NSPs through their fermented products may promote the growth of specific beneficial colonic bacteria which offer a prebiotic effect. Various modes of action of NSPs as therapeutic agent have been proposed in the present review. In addition, NSPs based films and coatings for packaging and wrapping are of commercial interest because they are compatible with several types of food products. However, much of the physiological and nutritional impact of NSPs and the mechanism involved is not fully understood and even the recommendation on the dose of different dietary NSPs intake among different age groups needs to be studied.

Phytate and phytase in fish nutrition.

Phytate formed during maturation of plant seeds and grains is a common constituent of plant-derived fish feed. Phytate-bound phosphorus (P) is not available to gastric or agastric fish. A major concern about the presence of phytate in the aquafeed is its negative effect on growth performance, nutrient and energy utilization, and mineral uptake. Bound phytate-P, can be effectively converted to available-P by phytase. During the last decade, phytase has been used by aqua feed industries to enhance the growth performance, nutrient utilization and bioavailability of macro and micro minerals in fish and also to reduce the P pollution into the aquatic environment. Phytase activity is highly dependent on the pH of the fish gut. Unlike mammals, fish are either gastric or agastric, and hence, the action of dietary phytase varies from species to species. In comparison to poultry and swine production, the use of phytase in fish feed is still in an unproven stage. This review discusses effects of phytate on fish, dephytinisation processes, phytase and pathway for phytate degradation, phytase production systems, mode of phytase application, bioefficacy of phytase, effects of phytase on growth performance, nutrient utilization and aquatic environment pollution, and optimum dosage of phytase in fish diets.

Poly-β-hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax

The bacterial storage polymer poly-β-hydroxybutyrate (PHB) has the potential to be used as an alternative anti-infective strategy for aquaculture rearing. In this research, the effects of (partially) replacing the feed of European sea bass juveniles with PHB were investigated. During a 6-week trial period, the PHB showed the ability to act as an energy source for the fish. This indicated that PHB was degraded and used during gastrointestinal passage. The gut pH decreased from 7.7 to 7.2 suggesting that the presence of PHB in the gut led to the increased production of (short-chain fatty) acids. The diets supplemented with 2% and 5% PHB (w/w) induced a gain of the initial fish weight with a factor 2.4 and 2.7, respectively, relative to a factor 2.2 in the normal feed treatment. Simultaneously, these treatments showed the highest bacterial range-weighted richness in the fish intestine. Based on molecular analysis, higher dietary PHB levels induced larger changes in the bacterial community composition. From our results, it seems that PHB can have a beneficial effect on fish growth performance and that the intestinal bacterial community structure may be closely related to this phenomenon.

The interactive effects of ammonia exposure, nutritional status and exercise on metabolic and physiological responses in gold fish (**Carassius auratus** L.)

This study aimed to elucidate the physiological effects of high environmental ammonia (HEA) following periods of feeding (2% body weight) and starvation (unfed for 7 days prior to sampling) in gold fish (Carassius auratus). Both groups of fish were exposed to HEA (1 mg/L; Flemish water quality guideline) for 0 h (control), 3 h, 12 h, 1 day, 4 days, 10 days, 21 days and 28 days. Measurements of weight gain (%), oxygen consumption (MO2), ammonia excretion rate, ammonia quotient (AQ), critical swimming speeds (Ucrit), plasma and muscle ammonia accumulation, plasma lactate, liver and muscle glycogen, lipid and protein content were done at various time intervals during the experimental periods. Overall, ammonia excretion rates, plasma ammonia accumulation and AQ were significantly affected by food regime in ammonia free water. HEA, the additional challenge in the present study, significantly altered all the studied parameters among fed and starved groups in days-dependent manner. Results show that weight gain (%), MO2, Ucrit, protein content in liver and muscle, and glycogen content in muscle among starved fish under HEA were considerably reduced compared to control and fed fish. Additionally a remarkable increase in plasma ammonia level, muscle ammonia, lactate accumulation and AQ was seen. However in fed fish, MO2, ammonia excretion rate, AQ and lactate level augmented after exposure to HEA. These results indicate that starved fish appeared more sensitive to HEA than fed fish. Furthermore, as expected, the toxic effect of ammonia exposure in both feeding treatments was exacerbated when imposed to exhaustive swimming (swum at 3/4th Ucrit). Such effects were more pronounced in starved fish. This suggests that starvation can instigate fish more vulnerable to external ammonia during exercise. Therefore, it was evident from our study that feeding ameliorates ammonia handling and reduces its toxicity during both routine and exhaustive swimming. Moreover, recovery was observed for some physiological parameters (e.g. MO2, ammonia excretion, Ucrit, plasma ammonia) during the last exposure periods (21-28 days) while for others (e.g. growth, tissue glycogen and protein content, muscle ammonia) effects only became apparent at this time. In the future, these results need to be considered in ecological context as fish in ammonia polluted may experience different phenomenon (starvation and exercise) simultaneously.

Anti-Oxidative Defences Are Modulated Differentially in Three Freshwater Teleosts in Response to Ammonia-Induced Oxidative Stress

Oxidative stress and the antioxidant response induced by high environmental ammonia (HEA) were investigated in the liver and gills of three freshwater teleosts differing in their sensitivities to ammonia. The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed to 1 mM ammonia (as NH4HCO3) for 0 h (control), 3 h, 12 h, 24 h, 48 h, 84 h and 180 h. Results show that HEA exposure increased ammonia accumulation significantly in the liver of all the three fish species from 24 h–48 h onwards which was associated with an increment in oxidative stress, evidenced by elevation of xanthine oxidase activity and levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Unlike in trout, H2O2 and MDA accumulation in carp and goldfish liver was restored to control levels (84 h–180 h); which was accompanied by a concomitant increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase activity and reduced ascorbate content. Many of these defence parameters remained unaffected in trout liver, while components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase) enhanced to a greater extent. The present findings suggest that trout rely mainly on glutathione dependent defensive mechanism while carp utilize SOD, CAT and ascorbate as anti-oxidative sentinels. Hepatic cells of goldfish appear to utilize each of these protective systems, and showed more effective anti-oxidative compensatory responses towards HEA than carp, while trout were least effective. The present work also indicates that HEA exposure resulted in a relatively mild oxidative stress in the gills of all three species. This probably explains the almost complete lack of anti-oxidative responses in branchial tissue. This research suggests that oxidative stress, as well as the antioxidant potential clearly differ between salmonid and cyprinid species.

Modulation of Rh glycoproteins, ammonia excretion and Na+ fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia

SUMMARY We investigated relationships among branchial unidirectional Na+ fluxes, ammonia excretion, urea excretion, plasma ammonia, plasma cortisol, and gill transporter expression and function in three freshwater fish differing in their sensitivity to high environmental ammonia (HEA). The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia-sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed chronically (12–168 h) to 1 mmol l−1 ammonia (as NH4HCO3; pH 7.9). During HEA exposure, carp and goldfish elevated ammonia excretion (JAmm) and Na+ influx rates () while trout experienced higher plasma ammonia (TAmm) and were only able to restore control rates of JAmm and . All three species exhibited increases in Na+ efflux rate (). At the molecular level, there was evidence for activation of a ‘Na+/NH4+ exchange metabolon’ probably in response to elevated plasma cortisol and TAmm, though surprisingly, some compensatory responses preceded molecular responses in all three species. Expression of Rhbg, Rhcg (Rhcg-a and Rhcg-b), H+-ATPase (V-type, B-subunit) and Na+/K+-ATPase (NKA) mRNA was upregulated in goldfish, Rhcg-a and NKA in carp, and Rhcg2, NHE-2 (Na+/H+ exchanger) and H+-ATPase in trout. Branchial H+-ATPase activity was elevated in goldfish and trout, and NKA activity in goldfish and carp, but NKA did not appear to function preferentially as a Na+/NH4+-ATPase in any species. Goldfish alone increased urea excretion rate during HEA, in concert with elevated urea transporter mRNA expression in gills. Overall, goldfish showed more effective compensatory responses towards HEA than carp, while trout were least effective.

Combined effects of high environmental ammonia, starvation and exercise on hormonal and ion-regulatory response in goldfish (Carassius auratus L.).

Due to eutrophication, high environmental ammonia (HEA) has become a frequent problem in aquatic environments, especially in agricultural or densely populated areas. During certain periods, e.g. winter, feed deprivation may occur simultaneously in natural waters. Additionally, under such stressful circumstances, fish may be enforced to swim at a high speed in order to catch prey, avoid predators and so on. Consequently, fish need to cope with all these stressors by altering physiological processes which in turn are controlled by genes expression. Therefore, in the present study, ammonia toxicity was tested in function of nutrient status (fed versus starved) and swimming performance activity (routine versus exhaustive). Goldfish, a relatively tolerant cyprinid, were exposed to HEA (1 mg/L; Flemish water quality guideline for surface water) for a period of 3 h, 12 h, 1 day, 4 days, 10 days, 21 days and 28 days and were either fed (2% body weight) or starved (kept unfed for 7 days prior to sampling). Results showed that the activity of Na⁺/K⁺-ATPase in the gills was stimulated by HEA and disturbance in ion balance was obvious with increases in plasma [Na⁺], [Cl⁻] and [Ca²⁺] after prolonged exposure. Additionally, osmoregulation and metabolism controlling hormones like cortisol and thyroid hormones (T3 and T4) were investigated to understand adaptive responses. The expression kinetics of growth, stress and osmo-regulatory representative genes such as Insulin-like growth factor 1 (IGF-I), growth hormone receptor (GHR), thyroid hormone receptor β (THRβ), prolactin receptor (PRLR), cortisol receptor (CR) and Na⁺/K⁺-ATPase α(3) were examined. Overall effect of HEA was evident since Na⁺/K⁺-ATPase activity, plasma cortisol, Na⁺ and Ca²⁺ concentration, expression level of CR and Na⁺/K⁺-ATPase α₃ mRNA in fed and starved fish were increased. On the contrary, transcript level of PRLR was reduced after 4 days of HEA; additionally T3 level and expression of GHR, IGF-I and THRβ genes were decreased following 10-21 days of HEA. Starvation, the additional challenge in the present study, significantly increased plasma cortisol level and CR transcript level under HEA compared to the fed exposed and control fish. Furthermore, a remarkable reduction in T3 and mRNA levels of THRβ, IGF-I and GHR genes was observed under starvation. The toxic effects in both feeding treatments were exacerbated when imposed to exhaustive swimming with more pronounced effects in starved fish. This confirms that starvation makes fish more vulnerable to external ammonia, especially during exercise.

Differential responses in ammonia excretion, sodium fluxes and gill permeability explain different sensitivities to acute high environmental ammonia in three freshwater teleosts.

We examined the acute physiological responses to high environmental ammonia (HEA), particularly the linkages between branchial ammonia fluxes and unidirectional Na(+) fluxes, as well as urea excretion, cortisol, and indicators of gill permeability in three freshwater teleosts differing in their sensitivities to ammonia; the highly sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less sensitive cyprinid Cyprinus carpio (common carp) and the highly resistant cyprinid Carassius auratus (goldfish). Fish were acutely exposed to two sub-lethal ammonia concentrations (as NH(4)HCO(3)) at pH 7.9: 1 mM for a period of 12 h, identical for all species, and 5 mM for the cyprinids and 1.4 mM for the trout for 3 h. Elevation of plasma cortisol at both levels of HEA was apparent in all species. At 1 mM, ammonia excretion (J(amm)) was inhibited to a greater extent in trout than cyprinids and concurrently a significantly higher plasma ammonia level was evident in trout. However J(amm) was reversed in all species at 5 or 1.4 mM. Goldfish showed a significant increase in urea excretion rate (J(urea)) during HEA exposure. In carp and trout, neither level of HEA elevated J(urea) but urea production was increased as evidenced by a considerable elevation of plasma urea. At 1mM HEA, Na(+) imbalance became progressively more severe in trout and carp due to a stimulation of unidirectional Na(+) efflux (J(out)(Na)) without a concomitant increase in unidirectional Na(+) influx (J(in)(Na)). Additionally, a transient reduction of J(in)(Na) was evident in trout. Goldfish showed an opposite trend for J(out)(Na) with reduced efflux rates and a positive Na(+) balance during the first few hours of HEA. However, after 12 h of exposure, both J(in)(Na) and J(out)(Na) were also increased in both carp and goldfish, whereas only J(out)(Na) was increased in trout, leading to a net Na(+) loss. Na(+) homeostasis was entirely disrupted in all three species when subjected to the 5 or 1.4 mM ammonia for 3 h: J(in)(Na) was significantly inhibited while considerable activation of J(out)(Na) was observed. Diffusive water efflux rates and net K(+) loss rates across the gills were enhanced during HEA only in trout, indicating an increment in gill transcellular permeability. Transepithelial potential was increased in all the species during ammonia exposure, but to the least extent in goldfish. Overall, for several different physiological systems, trout were most disturbed, and goldfish were least disturbed by HEA, helping to explain the differential ammonia tolerance of the three species.

Metal accumulation and metallothionein induction in the spotted dogfish **Scyliorhinus canicula**

Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10microM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587microg/L), Cd (1124microg/L), Pb (2072microg/L), Cu (635microg/L), and Ag (1079microg/L and two additional exposures at 10microg/L and 1microg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.

Linking environmental heavy metal concentrations and salinity gradients with metal accumulation and their effects: A case study in 3 mussel species of Vitória estuary and Espírito Santo bay, Southeast Brazil.

The present study was conducted to link the heavy metal load in three species of mussels (Perna perna, Mytella falcata and Mytella guyanensis) from the estuaries and bays around Vitória island, south-east of Brazil, with the salinity gradient and the heavy metal levels in the abiotic environment (including water, suspended particulate matter (SPM) and sediment). Primarily based on the salinity gradient, a total of 26 sites around Vitória Island were selected for sampling of water, SPM, sediments and organisms. Besides tissue metal levels, the condition index and energy stores (glycogen, lipid and protein) were quantified as an indicator of fitness in response to metal pollution. Dissolved metals in water indicate that Cd and Mn content was higher along Espírito Santo Bay, while Al, Co, Cu, Cr and Fe were elevated in the sites with low salinity such as river mouths, estuarine and sewage canals. Likewise, suspended matter sampled from low salinity sites showed a higher heavy metal load compared to moderate and high salinity sites. Though mussels were sampled from different sites, the contamination for Cd, Cu, Fe and Mn was higher in mussels inhabiting low salinity sites (M. guyanensis and M. falcata) compared to P. perna, a high saline water inhabitant. However, a higher Zn body burden was observed for P. perna compared to Mytella species. Tissue Fe accumulation (but not Mn and Zn) correlated with heavy metal levels in suspended material for all three species, and for M. falcata this correlation also existed for Cd and Cu. Energy store and condition index in all mussels varied depending on the sampling sites and correlated with salinity gradient rather than tissue metal concentration. Overall, metal concentration in mussels did not exceed the safe levels as per the international standards for metals, and would be of no risk for human consumption.