Comparative efficacy of phytase from fish gut bacteria and a commercially available phytase in improving the nutritive value of sesame oilseed meal in formulated diets for fingerlings of rohu, Labeo rohita (Actinopterygii: Cypriniformes: Cyprinidae)

Background. Phytate (myo-inositol 1,2,3,4,5,6-hexakis-dihydrogen phosphate) is the main storage form of phosphorus (P) and up to 80% of the total P content in plants remains unavailable to fi sh due to lack of intestinal phytases for effi cient phytate hydrolysis. The inclusion of microbial phytase in the feed is an approach to increase phytate phosphorus bioavailability. In the presently reported study, a comparison of the effi cacy of phytase produced by fi sh gut bacterial strain, Bacillus licheniformis LF1 and a commercially available phytase, BiophosTS in improving the nutritive value of sesame (Sesamum indicum) oilseed meal (SOM) was evaluated in the diet for fi ngerlings of rohu, Labeo rohita (Hamilton, 1822). Materials and methods. Eleven isonitrogenous (approximately 35% crude protein) and isocaloric (17.58 kJ · g–1 gross energy) experimental diets were formulated with the same basal diet containing raw sesame oilseed meal (D1) or oilseed meal pretreated with phytase from the fi sh gut bacterial strain, B. licheniformis LF1 (D2–D6) and commercially available phytase, Biophos-TS (D7–D11) at 10, 20, 30, 40, and 50 FTU · kg–1 and fed to rohu fi ngerlings (mean initial weight 1.85 ± 0.52 g) for 80 days. Results. Fermentation of oilseed meal signifi cantly reduced the crude fi bre content and anti-nutritional factors tannins and phytic acid, and enhanced mineral concentration. In terms of growth, feed conversion ratio, and protein effi ciency ratio, diets containing sesame oilseed meal pretreated with Biophos-TS at a concentration of 30 FTU · kg–1 and with strain LF1 at a concentration of 40 FTU · kg–1 resulted in a signifi cantly (P < 0.05) better performance of rohu fi ngerlings. The apparent digestibility of protein, lipid, ash and minerals was signifi cantly (P < 0.05) higher in fi sh fed diets D9 and D5 in comparison to those fed reference diet (RD). The crude protein, crude lipid, and ash contents of fi sh carcass were highest in fi sh fed diet D9, which was not signifi cantly (P < 0.05) different from those in the group of fi sh fed diet D5. Pretreatment of diets with phytase reduced faecal P levels. Conclusion. Comparison of the effi cacy of phytase produced by fi sh gut bacteria with commercially available phytase indicated no signifi cant difference in performance of rohu fi ngerlings in terms of growth and nutrient and mineral utilization.


INTRODUCTION
Unlike most domesticated farm animals, the majority of fi sh species generally require higher levels of dietary protein for optimum growth. High quality fi sh meal, with amino acid profi le that matched the fi sh's requirement pattern is therefore, commonly used at high levels. However, the concomitant rise in its price, uncertain availability, and fl uctuating quality have led to the search for alternative protein sources for fi sh feed to sustain fi sh production. Many leguminous oilseeds have been reported as feeds in fi sh culture, and the use of plant protein sources to completely or partially replace fi sh meal in fi sh diets has been reported in a number of studies (Hardy 2010, Nang Thu et al. 2011, Roy et al. 2014. From the results of these studies, it is quite clear that plant protein cannot be employed as the sole source of dietary protein, and the maximum recommended level of inclusion appears to be between 20% and 30% of the diet (Hardy and Barrows 2008). One of the major problems associated with the use of plant proteins in fi sh feed is the presence of antinutritional factors, which are endogenous compounds in feedstuffs that may reduce feed intake, growth, nutrient digestibility and utilization, affect the function of internal organs, or alter disease resistance. In plant feedstuffs, phytate (myo-inositol 1,2,3,4,5,6-hexakis-dihydrogen phosphate) is the main storage form of phosphorus (P) (Krogdahl et al. 2010, Hardy 2010) and up to 80% of the total P content in plants may be present in the form of phytate and is practically not available for monogastric or agastric aquatic animals (Anonymous 1993) due to lack of intestinal phytases for effi cient phytate hydrolysis (Nwanna and Schwarz 2007, Rao et al. 2009, Hardy 2010.
Phytase (E.C.3.1.3.8. myo-inositol hexaphosphate phosphohydrolase) is a hydrolytic enzyme that initiates the release of phosphate from phytic acid, which is the predominant form of phosphorus in cereal grains, oilseeds, and legumes (Cao et al. 2007, Huang et al. 2009). The presence of phytate in feed is undesirable as it chelates nutritionally important divalent cations like potassium, magnesium, zinc, iron, calcium, and copper, and proteins and amino acids, thereby rendering them biologically unavailable to the animal (Sardar et al. 2007, Afi nah et al. 2010, Krogdahl et al. 2010. Phytic acid in the form of phytate also inhibits digestive enzymes in fi sh (Khan and Ghosh 2013). Thus, the inclusion of plant proteins in fi sh diets may cause increased phosphorus discharge into the environment and a reduction in growth resulting from the decreased bioavailability of minerals (Cao et al. 2007(Cao et al. , 2008. The phytate phosphorus is excreted into the environment and is acted upon by microorganisms that release the phosphorus, causing pollution in terms of algal growth (Cao et al. 2007(Cao et al. , 2008. Roy et al. (2009) fi rst confi rmed the existence of phytase-producing bacteria in the gastrointestinal tracts of fi sh. The inclusion of microbial phytase in the feed is an approach to increase phytate phosphorus bioavailability (Sardar et al. 2007, Cao et al. 2008, Khan and Ghosh 2012. Das and Ghosh (2015) also reported effi cacy of sesame oil cake after bioprocessing through fermentation with a phytase-producing fi sh gut bacterium. Roy et al. (2014) assessed the role of phytase-producing fi sh gut bacteria in enhancing the nutritive value of sesame (Sesamum indicum) seed meal incorporated diets for rohu fi ngerlings and increasing the bioavailability of nutrients and minerals. There are several reports regarding the effi cacy of phytase on growth performance, body composition and phosphorus utilization by the fi sh fed diets pretreated with microbial phytase (Sugiura et al. 2001, Debnath et al. 2005, Baruah et al. 2007a, 2007b, Cao et al. 2008, Roy et al. 2014, Das and Ghosh 2015, but the report based on the comparison of the effi cacy of phytase produced by fi sh gut bacteria and commercially available phytase is lacking.
It is clear that supplemental phytase can enhance the digestibility and bio-availability of phosphorus, nitrogen and other minerals, reduce the amount of inorganic-P supplement to maximize growth and bone mineralization, and markedly decrease P load to aquatic environment (Cao et al. 2007, Morales et al. 2014, Yoo and Bai 2014. The optimum doses of phytase to replace inorganic P have not been evaluated in fi sh diets. Accordingly, further investigations about phytase application in fi sh feed are largely needed. In an earlier study in our laboratory with the phytase producing fi sh gut bacterial strain, Bacillus licheniformis LF1, we observed that this particular strain improved the nutritive value of sesame oilseed meal in the formulated diets for fi ngerlings of rohu, Labeo rohita (Hamilton, 1822) (see Roy et al. 2014). In the presently reported study, a comparative evaluation was done to assess the effi cacy of phytase produced by fi sh gut bacteria (Bacillus licheniformis LF1, isolated from the foregut region of L. rohita) and the commercially available phytase, Biophos-TS (Biochem Health Care, Mumbai, India, derived from fungus Aspergillus niger) in improving the nutritive value of sesame (Sesamum indicum) oilseed meal incorporated diets and increasing the bioavailability of nutrients and minerals like P, Ca, Mn, Fe, and Cu in the Indian major carp, rohu, Labeo rohita fi ngerlings through feeding trial.

MATERIALS AND METHODS
Microorganism used. The phytase-producing bacteria, Bacillus licheniformis LF1 (GenBank Accession Number: DQ351932) isolated from the proximal intestine of the Indian major carp, rohu, Labeo rohita (see Roy et al. 2009) was used for fermentation of sesame seed meal. Preparation of bacterial culture and fermentation of oilseed meal. The phytase-producing bacterial strain B. licheniformis LF1 was cultured in shake bottles in 4% tryptone soya broth (Hi-Media, Mumbai, India) for seed culture at 37 ± 1ºC for 24 h to obtain a mean viable count of 10 7 cells · mL -1 broth. This seed culture was transferred to the sterilized modifi ed phytase screening medium with optimum carbon, nitrogen, and vitamin sources and incubated under optimized culture conditions for the isolate LF1 as described by Roy et al. (2013). After 72 h of incubation, the bacterial culture was centrifuged at 10 000 × g for 10 min and the bacterial cells were discarded. The phytase activity of the culture supernatant was determined following Engelen et al. (1994) and the Fomazine Turbidity Unit (FTU) per mL of bacterial enzyme was calculated [1 FTU is defi ned as the amount of phytase that liberates 1μmol of inorganic phosphorus from 0.0051 mol · L -1 of sodium phytate per min at 37ºC and pH 5.5]. The enzyme extract was stored at -20ºC until further use. The sesame oilseed meals were sundried, fi nely ground, and passed through a fi ne meshed sieve to ensure homogeneity. The oilseed meal was sterilized by autoclaving and fermented in vitro with the bacterial phytase and commercially available phytase, Biophos-TS (Biochem Health Care Limited, Mumbai, India) at different concentrations (10,20,30,40, and 50 FTU · kg -1 ) at 37 ± 1ºC for 24 h prior to their incorporation into fi sh diets. The proximate composition, anti-nutritional factors (tannin and phytic acid) and mineral concentration of sesame oilseed meal were determined both before and after fermentation (Tables 1 and 2).

Experimental diets.
Eleven isonitrogenous (35% crude protein approximately) and isocaloric (17.58 kJ · g -1 gross energy approximately) experimental diets were prepared with a basal diet containing raw sesame oilseed meal (D1), experimental diets (D2-D6) containing sesame oilseed meal pretreated with bacterial phytase at different concentrations (D2 = 10 FTU · kg -1 ; D3 = 20 FTU · kg -1 ; D4 = 30 FTU · kg -1 ; D5 = 40 FTU · kg -1 ; D6 = 50 FTU · kg -1 ) and diets D7 through D11, containing sesame oilseed meal treated with commercially available phytase, Biophos-TS at different concentrations (D7 = 10 FTU · kg -1 ; D8 = 20 FTU · kg -1 ; D9 = 30 FTU · kg -1 ; D10 = 40 FTU · kg -1 ; D11 = 50 FTU · kg -1 ). The ingredient compositions of the experimental diets are given in Table 3. A diet containing fi sh meal as the main protein source was used as the reference diet (RD). To each of the formulated diet 1% chromic oxide was added as digestibility marker. The diets were prepared in pelleted form using 0.5% carboxymethylcellulose as binder and the pellets were dried in oven and stored in airtight containers in a refrigerator at 4ºC until used. The proximate composition of experimental diets is presented in Table 4. Experimental design. The feeding trial was conducted in fl ow-through 90 L circular fi bre-glass tanks for 80 days under laboratory conditions. Each tank was supplemented with unchlorinated water from a deep tube well with continuous aeration. Rohu, Labeo rohita, fi ngerlings were obtained from a local fi sh seed dealer and acclimatized for 15 days and fed with a mixture of rice bran and mustard oil cake. The fi ngerlings (mean initial weight 1.85 ± 0.52 g) were randomly distributed in the fi bre-glass tanks at a stocking density of 15 fi sh per tank with three replicates for each dietary treatment. Fish were fed once daily at 1000 h at a fi xed feeding rate of 3% of total body weight per day. To determine the feed consumption, any leftover feed was collected 6 h after each feeding and weighed after oven drying. The digestibility study was conducted separately in static aquaria. The faecal samples were collected everyday in the morning by siphoning 17 h after removal of the uneaten feed following the "immediate pipetting" method outlined by Spyridakis et al. (1989) from three replicates of each dietary treatment. The faeces naturally released by the fi sh could be easily detected and were immediately removed from the water with a glass canula. Pooled samples of uneaten feed and faecal matters for each dietary treatment were dried at 55°C and stored in a refrigerator for subsequent analysis. At the termination of 80-day experiment the fi sh were weighed and analysed for carcass composition. There was no mortality of fi sh during the feeding trial. The water quality parameters from each tank were monitored each week throughout the experimental period for temperature, pH, dissolved oxygen, and total alkalinity. The ranges of water quality parameters were: temperature, 29-32°C; pH, 7-7.8; dissolved oxygen, 4.6-5.5 mg · L -1 ; and alkalinity, 155-170 mg · L -1 . Chemical analyses and data collection. Feed ingredients, experimental diets, faecal samples, and fi sh carcass (at the beginning and after the termination of experiment) were analysed for proximate composition according to AOAC procedures (Anonymous 1990) and the procedures as described by Roy et al. (2014). Tannin and phytic acid contents in both fermented and raw sesame oilseed meal were determined following Schanderi (1970) and Wheeler and Ferrel (1971), respectively. Phosphorus and calcium contents in the diets, faecal samples, and carcass were estimated following Fiske and Subba Row as described by Hawk (1960) and Clark-Collip modifi cation of Kramer-Trisdall method as described by Hawk (1960), respectively. Mn, Cu, and Fe contents were estimated using an atomic absorption spectrophotometer (Electronics Corporation of India,    Hyderabad, Andhra Pradesh, India, Model: AAS4129). The intestinal phytase activity was estimated following the method of Engelen et al. (1994). One phytase unit (U) was defi ned as the amount of enzyme per mL of culture fi ltrate that released 1 μg of inorganic phosphorus per min. The water quality parameters were monitored following the methods outlined by APHA (Anonymous 1985).
Fish performance in terms of weight gain [%], specifi c growth rate (SGR) [% · day -1 ], feed conversion ratio (FCR), protein effi ciency ratio (PER), and apparent net protein utilization (ANPU) was determined using the standard formulae (Steffens 1989). Statistical analysis. Statistical analysis was done by one-way analysis of variance (ANOVA) using MS-Excel software (Microsoft Offi ce 2007). Mean difference between treatments were tested for signifi cance at P < 0.05 and comparisons were made by Duncan's multiple range test (Duncan 1955) to fi nd out which treatment differed signifi cantly from the other in respect of growth, carcass composition, digestibility, and general performance of the fi sh.

Effect of fermentation of sesame seed meal on proximate composition, anti-nutritional factors, and mineral concentration.
Fermentation with the phytase from the fi sh gut bacterial strain Bacillus licheniformis LF1 and commercially available phytase, Biophos-TS resulted in reduction of anti-nutritional factors, increase in crude protein and crude lipid levels ( Table 1). The level of crude protein in fermented sesame oil seed meal increased from 22.86% to 25.65% for LF1 (40 FTU · kg -1 ) and 25.84% for Biophos-TS (30 FTU · kg -1 ), whereas, crude lipid level increased from 5.15% to 6.00% for LF1 (30 FTU · kg -1 ) and 6.15% for Biophos-TS (30 FTU · kg -1 ). In both the cases, the level of free amino acids increased from 0.27% to 0.52%. Fermentation of sesame oil seed meal also resulted in signifi cant increase in its mineral contents (Table 2). Fish growth performance and feed utilization. The growth performance and feed utilization of rohu fi ngerlings are depicted in Table 5. The mean fi nal weight of the fi sh increased signifi cantly from the initial value in all dietary treatments. Fish reared on diet D9 (containing sesame oilseed meal fermented with Biophos-TS at a concentration of 30 FTU · kg -1 ) and D5 (containing sesame oilseed meal fermented with LF1 strain at a concentration of 40 FTU · kg -1 ) showed good performance in terms of live weight gain (%), specifi c growth rate (SGR), protein effi ciency ratio (PER), and apparent net protein utilization (ANPU). However, live weight gain (%) and specifi c growth rate (SGR), were not signifi cantly (P < 0.05) different from the group of fi sh fed diet D6 (containing oilseed meal fermented with LF1 strain at a concentration of 50 FTU · kg -1 ) and D10 (containing oilseed meal fermented with Biophos-TS at a concentration of 40 FTU · kg -1 ). Feed conversion ratio (FCR) was lowest for diet D9 which was not signifi cantly (P < 0.05) different from that for the fi sh fed diet D5 and D10, FCR was highest for the groups of fi sh fed diet D1 that was not signifi cantly (P < 0.05) different from that for the groups of fi sh fed diets RD and D2.
Apparent nutrient and mineral digestibility. The apparent digestibility of protein, lipid, and ash were better in fi sh fed diets D9 and D5, which were not signifi cantly (P < 0.05) different in the group of fi sh fed diets D6, D10, and D11. Maximum dry matter digestibility was recorded in fi sh fed diet D2 and was not signifi cantly different from that in the fi sh fed reference diet (Fig. 1). Maximum phosphorus, calcium, manganese, copper, and iron digestibility was recorded in the group of fi sh fed diet D9, which was not signifi cantly (P < 0.05) different in the groups of fi sh fed diets D5, D6, and D10 (Figs. 2a and b). Proximate carcass composition. Proximate carcass composition in experimental fi sh at the termination of the feeding experiment is depicted in Fig. 3. The deposition of carcass protein and lipid was signifi cantly (P < 0.05) maximal in fi sh fed fermented sesame seed meal incorporated diets than the reference diet. The crude protein, crude lipid, and ash contents of fi sh carcass were highest in fi sh fed diet D9, which was not signifi cantly (P < 0.05) different from those in the group of fi sh fed diet D5. Mineral concentration in the carcass of experimental fi sh increased over the initial value in all dietary treatments and an increasing level of phytase was associated with an increase in carcass mineral content ( Table 6). The phosphorus, calcium, manganese, copper, and iron contents in the fi sh carcass were highest for diet D9, which were not signifi cantly (P < 0.05) different from those in the group of fi sh fed diet D5. In case of calcium, no signifi cant (P < 0.05) difference was noticed in the fi sh fed diet D6 (containing sesame oilseed meal fermented with bacterial strain LF1 at a concentration of 50 FTU · kg -1 ) and D10 (containing sesame oilseed meal fermented with commercially Biophos-TS at a concentration of 40 FTU · kg -1 ). Intestinal phytase activity. Intestinal phytase activity in experimental fi sh at the termination of the feeding experiment is presented in Fig. 4. The intestinal phytase activity increased with increasing level of incorporation of bacterial or commercial phytase in the diets. Intestinal phytase activity was higher in the groups of fi sh fed fermented oilseed meal incorporated diets in comparison to that fed raw seed meal incorporated diet. The highest phytase activity was recorded in the group of fi sh fed diet D9 which was not signifi cantly different from that in the groups of fi sh fed experimental diets D5 and D11. Faecal phosphorus concentration. The faecal phosphorus concentration was signifi cantly higher in fi sh fed (Fig. 5) raw sesame seed meal incorporated diet than the reference diet and an increasing level of phytase in the diets was associated with a decrease in faecal phosphorus concentration. Maximum faecal phosphorus concentration was detected in the fi sh fed diet D1, containing raw sesame oilseed meal.

DISCUSSION
In the presently reported investigation small fi ngerlings (mean initial weight 1.85 ± 0.52 g) of Labeo rohita were used to evaluate dietary effect on the growth of the fi sh. Small fi sh respond faster than large fi sh to nutritional variables. Moreover, small fi sh are more sensitive to diet differences; if small fi sh are unaffected, it is a safe assumption that larger fi sh will not be (Lovell 1998). The results of our study indicated that the mean fi nal weight of the fi sh increased considerably from the initial value in all dietary treatments. The overall growth performance of rohu fi ngerlings, fed fermented sesame oilseed meal, incorporated diets in both cases were better in comparison to fi sh fed raw oilseed meal diet. The observation on weight gain in both cases i.e., phytase from fi sh gut bacteria Bacillus licheniformis LF1 and commercially available phytase Biophos-TS corroborates the fi ndings of Liebert and Portz (2005), who reported that different sources of microbial phytase similarly affected the growth in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758). The differences in the growth performance recorded in this study agree with the report of Nwanna et al. (2005) Debnath et al. 2005). In L. rohita also growth performance was signifi cantly improved on corn gluten meal (30%) based experimental diets with graded levels of phytase pre-treatment (Hussain et al. 2011). However, phytase pre-treatment did not improve the growth of rainbow trout fed diets containing canola protein concentrate (Forster et al. 1999), juvenile Korean rock fi sh, Sebastes schlegeli Hilgendorf, 1880, fed diets containing soybean meal (Yoo et al. 2005) and Atlantic salmon, Salmo salar Linnaeus, 1758, fed soy protein concentrate treated with graded levels of phytase (Carter and Sajjadi 2011). Nwanna and Schwarz (2007) also found only marginal effects on the mean weight gain, specifi c growth rate, and feed conversion ratio when common carp was fed with phytase pre-treated diet. This discrepancy may be due to the differences in the phytase pretreatment methods of the feedstuff and also different rearing conditions. Hauler and Carter (1997) reported that phytase stimulated appetite which increased the growth of Atlantic salmon, S. salar. In channel catfi sh, Ictalurus punctatus (Rafi nesque, 1818), also phytase pretreatment stimulated appetite and increased the growth through increased feed intake (Li and Robinson 1997). Formation of complex between sesame seed α-globulin and sodium phytate has been described as a bi-phasic reaction (Rajendran and Prakash 1993). Initially phytase binds protein through strong electrostatic attractions which is followed by slower protein-protein interactions resulting in precipitation when the protein-phytate complex exceeds a critical size. It appears that phytase Values are mean ± standard error of the mean (n = 3); Sesame diet = diet with raw sesame seed meal; Experimental diets and respective phytase content [FTU · kg  Values are mean ± standard error of the mean ( (both bacterial and commercial) in the presently reported study partially prevented the formation of protein-phytate complexes by prior hydrolysis of phytate to release protein so that it becomes available for growth. In the presently reported, improvement in growth performance of rohu fi ngerlings may be attributed to enhanced release of nutrients by breaking down the bonds between phytateprotein complex. Improvement in growth performance of rohu fi ngerlings may also be attributed to the improved use of phytate-P as well as phytate-bound protein.
Feeding rate is important for the growth, feed conversion, nutrient retention effi ciency, and chemical composition of fi sh (Hung and Lutes 1987). An optimum feeding rate is helpful to minimize the feed loss, reduce water pollution, and decrease cost of aquaculture production (Du et al. 2006). In earlier experiments in this laboratory, it was established that rohu fi ngerlings fed to apparent satiation displayed a feed intake more uniform and close to 3% BW · day -1 fi xed feeding level (Bairagi et al. 2002, 2004, Ramachandran and Ray, 2007, Roy et al. 2014. Therefore, in the presently reported experiment also rohu fi ngerlings were fed at a fi xed feeding rate of 3% BW · d -1 . In our study, the overall growth performance of rohu fi ngerlings remained low compared to its growth in natural and farm conditions. A similar growth trend has also been reported with other nutritional experiments conducted on Indian major carps including rohu (Ravi and Devaraj 1991, Bairagi et al. 2002, 2004, Benkappa and Varghese 2003, Khan et al. 2012, Roy et al. 2014. As fi shes are poikilotherms, drastic change in their surrounding water temperature will infl uence their metabolic processes, behaviour, migration, growth, reproduction, and survival (Fry 1971, Pörtner 2001. A possible explanation for this lower growth rate could be that Indian major carps are sensitive to environmental conditions and do not attain maximum growth in a confi ned environment compared with other hardy species such as tilapia and common carp (Benkappa and Varghese 2003). Another possible reason of poor fi sh growth might be due to low appetite and low feed utilization (Islam 2002). Dietary protein is the primary source of nitrogen, which is the other enriching nutrient of major concern in aquaculture (Hardy 2010). In this experiment, an increase in body protein accretion and nutrient (crude protein, lipid) digestibility was noticed in rohu fi ngerlings fed Biophos-TS pre-treated diet, D9. The performance of fi sh reared on the control and raw oilseed meal incorporated diets were poor compared to fi sh reared on other experimental diets in terms of weight gain, PER, FCR, deposition of crude protein and lipid in the carcass, and apparent nutrient (crude protein, lipid) digestibility. However, the signifi cant increase in body protein accretion and nutrient (crude protein, lipid) digestibility recorded from this study disagree with the report of Yan et al. (2002), who demonstrated that weight gain and dietary protein utilization of channel catfi sh were not improved by phytase addition.
It has been shown that the addition of microbial phytase enhanced the availability of various minerals from the plant oilseed meals and improved their absorption. Mineral concentration of the carcass of experimental fi sh increased over the initial value in all dietary treatments. Although the deposition of phosphorus, manganese, copper, and iron in fi sh carcass was higher in rohu fi ngerlings fed Biophos-TS pretreated diet D9, they were not signifi cantly different from those in the fi sh fed bacterial phytase pretreated diet D5. In the case of calcium deposition, there was no signifi cant difference among the fi sh fed diets D5, D6, D9, and D10. Biophos-TS pretreated diets showed satisfactory results in terms of phosphorus-, calcium-, manganese-, copper-, and iron digestibility which was not signifi cantly different from those in the fi sh fed bacterial phytase pretreated diet.   Yoo and Bai 2014). Apparent digestibility of phosphorus, calcium, manganese, iron, and zinc from barley, canola meal, wheat, and wheat middling by rainbow trout also has been reported to increase with the addition of phytase (Gatlin and Li 2008). Omnivorous fi sh species such as channel catfi sh, Ictalurus punctatus, has been shown to exhibit increased phosphorus utilization from soybean meal based diets with the supplementation of phytase (Yan et al. 2002).

Experimental diets
In the presently reported experiment, pH of the experimental diets was maintained at 5.5 by adding citric acid. Several studies reported that microbial phytase has two optimal peaks of activity, one at pH 5.0-5.5 and the other at pH 2.5 (Simons et al. 1990). On the other hand, phytase activity changes along the digestive tract (Yi and Kornegay 1996). There are some evidences that pH of the gastrointestinal tract affects the bioavailability of minerals in fi sh (Sugiura et al. 1998, Vielma et al. 1999). In addition to their effect on intestinal pH, supplementary organic acids can also bind various cations along the intestine (Ravindran and Kornegay 1993), resulting in increased intestinal absorption of minerals (Sugiura et al. 1998, Vielma et al. 1999. Citric acid has been reported to increase P bioavailability by dephosphorylation of phytate in vitro (Żyła et al. 1995). Baruah et al. (2007aBaruah et al. ( , 2007b and Sugiura et al. (2001) also reported that addition of citric acid into the fi sh feed increased P utilization in Labeo rohita juveniles and also apparent absorption of Mg and P in Oncorhynchus mykiss, respectively. In carnivorous fi sh like rainbow trout, production of acids assist in lowering the dietary pH, but in agastric species like L. rohita, no such mechanism exists. Hence, addition of organic acids or acidifi ers may reduce the dietary pH, which in turn may reduce the pH of the intestine and enhance mineral absorption. Further, dietary acidifi cation may reduce the rate of gastric emptying (Mayer 1994), which may favour the action of phytase. Thus, it was hypothesized that dietary addition of organic acids and microbial phytase has a synergistic effect on mineral availability. In the presently reported study, the signifi cant increase in growth parameters, body protein accretion, nutrient (crude protein, lipid), and mineral digestibility may be due to the synergistic effect of dietary addition of organic acid (citric acid) and microbial phytase. Study of digestive enzymes is an essential step towards understanding the mechanism of digestion and how the organism adapts to changes in the nutritional environment (Xue et al. 1999). The gut (endogenous) phytase enzyme is the indicator of digestibility and utilization of nutrients and minerals. In this experiment, intestinal phytase activity was found to increase with increasing level of incorporation of phytase. In the presently reported study, the endogenous phytase activity was estimated in the fi sh intestine fed phytase pre-treated diets in order to compare the activity among the groups of fi sh fed bacterial and commercial phytase. Therefore, endogenous phytase activity was not estimated at initial stage as well as in the group of fi sh fed RD. Intestinal phytase activity was higher in the group of fi sh fed phytase pretreated diets in comparison to those fed on raw oilseed meal incorporated diets. It therefore, appears that pretreatment i.e., fermentation might have played an important role in enhancement of intestinal phytase activity.
In the presently reported study, the faecal phosphorus concentration was signifi cantly higher in fi sh fed raw sesame oilseed meal incorporated diets than the reference diet and an increasing level of phytase pre-treated SOM was associated with a decrease in faecal phosphorus concentration. Maximum faecal phosphorus concentration was found in the fi sh fed diet D1. The results of the present study indicate that reduction of P wastes could be accomplished by the incorporation of phytase in diets due to improved utilization of dietary phytin P. These fi ndings are in agreement with the fi ndings of previous workers who reported that phytase pre-treatment increases the utilization of dietary nutrients and minerals and also reduces pollution in aquatic environment by decreasing excretion of phosphorus and protein through faeces (Nwanna and Schwarz 2007, Sardar et al. 2007, Laining et al. 2012, Liu et al. 2012.

CONCLUSION AND FUTURE PERSPECTIVES
In conclusion, these results support the hypothesis that phytase (Biophos-TS and from fi sh gut bacteria Bacillus licheniformis) pre-treated diets containing high levels of plant feedstuffs will reduce the excretion of waste P thereby resulting in reduced P effl uent from aquaculture facilities. On the other hand, pre-treatment of diets with phytase may not only enhance the utilization of phosphorus but also other nutrients including protein and amino acids. Comparison of the effi cacy of phytase produced by fi sh gut bacteria with commercially available phytase (Biophos-TS) indicated no signifi cant difference in terms of growth rate, proximate carcass composition, apparent nutrient and mineral digestibility. Thus, preparation of fi sh feed by fermented with phytase produced by fi sh gut bacteria and commercial phytase may be expected to provide both economic and environmental benefi ts through decreased expenditures on supplemental minerals and mineral outputs to the aquatic ecosystem. However, it is too early to recommend to the industry to use the phytase pre-treated SOM in formulation of aquafeeds. The costs involved in phytase pre-treatment have yet to be justifi ed. Further research is necessary in this direction to evaluate the effi cacy of the pre-treatment strategy and the economic benefi ts of utilizing these products in aquafeeds.