Tanami ROY, Suhas K. DAN, Goutam BANERJEE, Ankita NANDI, Pinki GHOSH, and Arun K. RAY

15 Pages·2016·0.37 MB·English

ACTA ICHTHYOLOGICA ET PISCATORIA (2016) 46 (1): 9–23 DOI: 10.3750/AIP2016.46.1.02
COMPARATIVE EFFICACY OF PHYTASE FROM FISH GUT BACTERIA AND A COMMER-
CIALLY AVAILABLE PHYTASE IN IMPROVING THE NUTRITIVE VALUE OF SESAME
OILSEED MEAL IN FORMULATED DIETS FOR FINGERLINGS OF ROHU, LABEO ROHITA
(ACTINOPTERYGII: CYPRINIFORMES: CYPRINIDAE)
Tanami ROY, Suhas K. DAN, Goutam BANERJEE, Ankita NANDI, Pinki GHOSH,
and Arun K. RAY*
Fisheries Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan-731235, West Bengal, India
Roy T., Dan S.K., Banerjee G., Nandi A., Ghosh P., Ray A.K. 2016. Comparative effi cacy of phytase from
fi sh gut bacteria and a commercially available phytase in improving the nutritive value of sesame
oilseed meal in formulated diets for fi ngerlings of rohu, Labeo rohita (Actinopterygii: Cypriniformes:
Cyprinidae). Acta Ichthyol. Piscat. 46 (1): 9–23.
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, Biophos-
TS 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.
Keywords: growth performance, mineral utilization, phytase, plant ingredient, aquafeed, Indian major carp
INTRODUCTION for alternative protein sources for fi sh feed to sustain fi sh
Unlike most domesticated farm animals, the majority production. Many leguminous oilseeds have been reported
of fi sh species generally require higher levels of dietary as feeds in fi sh culture, and the use of plant protein sources
protein for optimum growth. High quality fi sh meal, with to completely or partially replace fi sh meal in fi sh diets
amino acid profi le that matched the fi sh’s requirement has been reported in a number of studies (Hardy 2010,
pattern is therefore, commonly used at high levels. Nang Thu et al. 2011, Roy et al. 2014). From the results
However, the concomitant rise in its price, uncertain of these studies, it is quite clear that plant protein cannot
availability, and fl uctuating quality have led to the search be employed as the sole source of dietary protein, and the
* Correspondence: Prof. Arun Kumar Ray, Fisheries Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan-731235, West Bengal, India, phone:
([phone], e-mail: (AKR) [email protected], (TR) [email protected], (SKD) [email protected], (GB) [email protected], (AN)
[email protected], (PG) [email protected].
10 Roy et al.
maximum recommended level of inclusion appears to be to maximize growth and bone mineralization, and markedly
between 20% and 30% of the diet (Hardy and Barrows decrease P load to aquatic environment (Cao et al. 2007,
2008). One of the major problems associated with the Morales et al. 2014, Yoo and Bai 2014). The optimum doses
use of plant proteins in fi sh feed is the presence of anti- of phytase to replace inorganic P have not been evaluated in
nutritional factors, which are endogenous compounds in fi sh diets. Accordingly, further investigations about phytase
feedstuffs that may reduce feed intake, growth, nutrient application in fi sh feed are largely needed.
digestibility and utilization, affect the function of internal In an earlier study in our laboratory with the phytase
organs, or alter disease resistance. In plant feedstuffs, producing fi sh gut bacterial strain, Bacillus licheniformis
phytate (myo-inositol 1,2,3,4,5,6-hexakis-dihydrogen LF1, we observed that this particular strain improved the
phosphate) is the main storage form of phosphorus nutritive value of sesame oilseed meal in the formulated
(P) (Krogdahl et al. 2010, Hardy 2010) and up to 80% of diets for fi ngerlings of rohu, Labeo rohita (Hamilton, 1822)
the total P content in plants may be present in the form (see Roy et al. 2014). In the presently reported study, a
of phytate and is practically not available for monogastric comparative evaluation was done to assess the effi cacy of
or agastric aquatic animals (Anonymous 1993) due to phytase produced by fi sh gut bacteria (Bacillus licheniformis
lack of intestinal phytases for effi cient phytate hydrolysis LF1, isolated from the foregut region of L. rohita) and the
(Nwanna and Schwarz 2007, Rao et al. 2009, Hardy 2010). commercially available phytase, Biophos-TS (Biochem
Phytase (E.C.3.1.3.8. myo-inositol hexaphosphate Health Care, Mumbai, India, derived from fungus
phosphohydrolase) is a hydrolytic enzyme that initiates Aspergillus niger) in improving the nutritive value of
the release of phosphate from phytic acid, which is the sesame (Sesamum indicum) oilseed meal incorporated diets
predominant form of phosphorus in cereal grains, oilseeds, and increasing the bioavailability of nutrients and minerals
and legumes (Cao et al. 2007, Huang et al. 2009). The like P, Ca, Mn, Fe, and Cu in the Indian major carp, rohu,
presence of phytate in feed is undesirable as it chelates Labeo rohita fi ngerlings through feeding trial.
nutritionally important divalent cations like potassium,
magnesium, zinc, iron, calcium, and copper, and proteins MATERIALS AND METHODS
and amino acids, thereby rendering them biologically Microorganism used. The phytase-producing bacteria,
unavailable to the animal (Sardar et al. 2007, Afi nah et Bacillus licheniformis LF1 (GenBank Accession Number:
al. 2010, Krogdahl et al. 2010). Phytic acid in the form DQ351932) isolated from the proximal intestine of the
of phytate also inhibits digestive enzymes in fi sh (Khan Indian major carp, rohu, Labeo rohita (see Roy et al.
and Ghosh 2013). Thus, the inclusion of plant proteins 2009) was used for fermentation of sesame seed meal.
in fi sh diets may cause increased phosphorus discharge Preparation of bacterial culture and fermentation
into the environment and a reduction in growth resulting of oilseed meal. The phytase-producing bacterial strain
from the decreased bioavailability of minerals (Cao et al. B. licheniformis LF1 was cultured in shake bottles in 4%
2007, 2008). The phytate phosphorus is excreted into the tryptone soya broth (Hi-Media, Mumbai, India) for seed
environment and is acted upon by microorganisms that culture at 37 ± 1ºC for 24 h to obtain a mean viable count of
release the phosphorus, causing pollution in terms of algal 107 cells · mL–1 broth. This seed culture was transferred to the
growth (Cao et al. 2007, 2008). Roy et al. (2009) fi rst sterilized modifi ed phytase screening medium with optimum
confi rmed the existence of phytase-producing bacteria carbon, nitrogen, and vitamin sources and incubated under
in the gastrointestinal tracts of fi sh. The inclusion of optimized culture conditions for the isolate LF1 as described
microbial phytase in the feed is an approach to increase by Roy et al. (2013). After 72 h of incubation, the bacterial
phytate phosphorus bioavailability (Sardar et al. 2007, culture was centrifuged at 10 000 × g for 10 min and the
Cao et al. 2008, Khan and Ghosh 2012). Das and Ghosh bacterial cells were discarded. The phytase activity of the
(2015) also reported effi cacy of sesame oil cake after bio- culture supernatant was determined following Engelen et
processing through fermentation with a phytase-producing al. (1994) and the Fomazine Turbidity Unit (FTU) per mL
fi sh gut bacterium. Roy et al. (2014) assessed the role of bacterial enzyme was calculated [1 FTU is defi ned as
of phytase-producing fi sh gut bacteria in enhancing the the amount of phytase that liberates 1μmol of inorganic
nutritive value of sesame (Sesamum indicum) seed meal phosphorus from 0.0051 mol · L–1 of sodium phytate per
incorporated diets for rohu fi ngerlings and increasing min at 37ºC and pH 5.5]. The enzyme extract was stored
the bioavailability of nutrients and minerals. There at –20ºC until further use. The sesame oilseed meals
are several reports regarding the effi cacy of phytase on were sundried, fi nely ground, and passed through a fi ne
growth performance, body composition and phosphorus meshed sieve to ensure homogeneity. The oilseed meal
utilization by the fi sh fed diets pretreated with microbial was sterilized by autoclaving and fermented in vitro with
phytase (Sugiura et al. 2001, Debnath et al. 2005, Baruah the bacterial phytase and commercially available phytase,
et al. 2007a, 2007b, Cao et al. 2008, Roy et al. 2014, Das Biophos-TS (Biochem Health Care Limited, Mumbai,
and Ghosh 2015), but the report based on the comparison India) at different concentrations (10, 20, 30, 40, and 50
of the effi cacy of phytase produced by fi sh gut bacteria FTU · kg–1) at 37 ± 1ºC for 24 h prior to their incorporation
and commercially available phytase is lacking. into fi sh diets. The proximate composition, anti-nutritional
It is clear that supplemental phytase can enhance the factors (tannin and phytic acid) and mineral concentration
digestibility and bio-availability of phosphorus, nitrogen and of sesame oilseed meal were determined both before and
other minerals, reduce the amount of inorganic-P supplement after fermentation (Tables 1 and 2).
Effi cacy of phytase in rohu nutrition 11
Table 1Effect of fermentation with phytase from fi sh gut bacterial strain LF1 and commercial phytase Biophos-TS on the proximate composition and anti-nutritional factors of sesame oilseed meal (on % dry matter basis) Sesame oilseed meal fermented with LF1 phytase Sesame oilseed meal fermented with Biophos-TSParameterD1D2D3D4D5D6D7D8D9D10D11 Moisture2.92 ± 0.082.77 ± 0.082.54 ± 0.072.48 ± 0.072.32 ± 0.072.40 ± 0.072.92 ± 0.092.84 ± 0.082.70 ± 0.082.57 ± 0.072.41 ± 0.07Dry matter96.85 ± 2.7997.23 ± 2.8097.46 ± 2.8197.52 ± 2.8197.68 ± 2.8297.60 ± 2.8297.08 ± 2.8097.16 ± 2.8097.30 ± 2.8197.43 ± 2.8197.59 ± 2.81 n eoCrude Protein22.86 ± 0.6624.08 ± 0.6924.45 ± 0.7024.79 ± 0.7125.65 ± 0.7425.35 ± 0.7324.12 ± 0.6924.50 ± 0.7025.84 ± 0.7425.19 ± 0.7325.41 ± 0.72tiatimsCrude fi bre19.82 ± 0.5716.67 ± 0.4816.00 ± 0.4615.85 ± 0.4514.05 ± 0.4014.18 ± 0.4116.59 ± 0.4816.04 ± 0.4615.70 ± 0.4513.98 ± 0.4014.07 ± 0.40oixpomCrude lipid5.15 ± 0.155.75 ± 0.175.80 ± 0.176.00 ± 0.175.92 ± 0.175.90 ± 0.175.85 ± 0.175.90 ± 0.176.15 ± 0.185.98 ± 0.176.00 ± 0.17rPocAsh10.02 ± 0.2910.10 ± 0.299.87 ± 0.2810.09 ± 0.2911.00 ± 0.329.32 ± 0.2711.56 ± 0.3311.35 ± 0.3310.45 ± 0.39.75 ± 0.2810.86 ± 0.31NFE39.23 ± 1.1340.63 ± 1.1741.34 ± 1.1940.79 ± 1.1841.06 ± 1.1842.85 ± 1.2338.96 ± 1.1239.37 ± 1.1439.16 ± 1.1342.53 ± 1.2341.25 ± 1.19–1Gross energy [kJ· g]17.54 ± 0.1217.79 ± 0.1217.92 ± 0.1217.96 ± 0.1217.88 ± 0.1218.09 ± 0.1317.29 ± 0.1217.63 ± 0.1217.96 ± 0.1218.00 ± 0.1217.79 ± 0.120.31 ± 0.010.28 ± 0.010.26 ± 0.010.22 ± 0.010.24 ± 0.010.29 ± 0.010.26 ± 0.010.21 ± 0.010.20 ± 0.010.18 ± 0.01Tannin1.97 ± 0.10 (84.3)(85.7)(86.8)(88.8)(87.8)(85.3)(86.8)(89.3)(89.8)(90.8)FN0.30 ± 0.010.23 ± 0.010.15 ± 0.010.09 ± 0.010.09 ± 0.010.27 ± 0.010.22 ± 0.010.10 ± 0.010.09 ± 0.010.09 ± 0.01APhytic acid1.68 ± 0.08(82.1)(86.3)(91.1)(94.6)(94.6)(83.9)(88.8)(94.0)(94.6)(94.6) –1Values are mean ± standard error of the mean (n = 3); Values in parentheses indicate percent reduction; Experimental diets and respective phytase content [FTU · kg]: D1 = raw sesame oilseed meal, D2 = 10, –1D3 = 20, D4 = 30, D5 = 40; D6 = 50, D7 = 10, D8 = 20, D9 = 30, D10 = 40, D11 = 50; [1 FTU is defi ned as the amount of phytase that liberates 1 μmol of inorganic phosphorus from 0.0051 mol · L of sodium phytate per min at 37ºC and pH 5.5]; NFE = nitrogen-free extract; ANF = anti-nutritional factor. Table 2Effect of fermentation with phytase from fi sh gut bacterial strain LF1 and commercial phytase, Biophos-TS on mineral content of sesame oilseed meal Raw sesame Sesame oilseed meal fermented by LF1phytase Sesame oilseed meal fermented by Biophos-TS Content of mineralsoilseed mealD2D3D4D5D6D7D8D9D10D11D1 12.04 ± 0.3512.85 ± 0.3713.24 ± 0.3814.00 ± 0.4013.96 ± 0.4012.24 ± 0.3512.96 ± 0.3714.09 ± 0.4113.76 ± 0.3913.65 ± 0.39–1]8.66 ± 0.25Phosphorus [mg · g(39.0)(48.4)(52.8)(61.6)(61.2)(41.3)(49.6)(62.7)(58.89)(57.62)0.94 ± 0.021.06 ± 0.0291.11 ± 0.0321.28 ± 0.0351.26 ± 0.0370.98 ± 0.0291.10 ± 0.0321.34 ± 0.0401.28 ± 0.0351.29 ± 0.037–1Calcium [mg · g]0.76 ± 0.02(23.68)(39.47)(46.05)(68.42)(65.78)(28.94)(44.73)(76.31)(68.42)(69.73)9.80 ± 0.2810.00 ± 0.2910.42 ± 0.3010.76 ± 0.3110.82 ± 0.3110.02 ± 0.2910.14 ± 0.2911.00 ± 0.3210.96 ± 0.3211.09 ± 0.32–1Iron [μg · g] 8.62 ± 0.25(13.68)(16.0)(20.88)(24.82)(25.52)(16.24)(17.63)(27.61)(27.14)(28.65)13.00 ± 0.3713.89 ± 0.4014.02 ± 0.4014.10 ± 0.4113.99 ± 0.4013.16 ± 0.3813.79 ± 0.3914.24 ± 0.4114.00 ± 0.4014.00 ± 0.40–1Manganese [μg · g]11.76 ± 0.34(10.54)(18.11)(20.5)(19.8)(18.9)(11.9)(17.26)(21.08)(19.0)(19.0)13.16 ± 0.3813.97 ± 0.4014.16 ± 0.4114.46 ± 0.4114.23 ± 0.4113.76 ± 0.3914.15 ± 0.4114.36 ± 0.4114.54 ± 0.4214.28 ± 0.41–1Copper [μg · g]10.75 ± 0.31(22.41)(29.95)(31.72)(34.51)(32.37)(28.0)(34.88)(33.5)(35.25)(32.8) See footnote to Table 1.
12 Roy et al.
Experimental diets. Eleven isonitrogenous (35% crude after oven drying. The digestibility study was conducted
protein approximately) and isocaloric (17.58 kJ · g–1 gross separately in static aquaria. The faecal samples were
energy approximately) experimental diets were prepared collected everyday in the morning by siphoning 17 h after
with a basal diet containing raw sesame oilseed meal removal of the uneaten feed following the “immediate
(D1), experimental diets (D2–D6) containing sesame pipetting” method outlined by Spyridakis et al. (1989)
oilseed meal pretreated with bacterial phytase at different from three replicates of each dietary treatment. The faeces
concentrations (D2 = 10 FTU · kg–1; D3 = 20 FTU · kg–1; naturally released by the fi sh could be easily detected and
D4 = 30 FTU · kg–1; D5 = 40 FTU · kg–1; D6 = 50 FTU · were immediately removed from the water with a glass
kg–1) and diets D7 through D11, containing sesame oilseed canula. Pooled samples of uneaten feed and faecal matters
meal treated with commercially available phytase, for each dietary treatment were dried at 55°C and stored in
Biophos-TS at different concentrations (D7 = 10 FTU · kg– a refrigerator for subsequent analysis. At the termination
1; D8 = 20 FTU · kg–1; D9 = 30 FTU · kg–1; D10 = 40 FTU · of 80-day experiment the fi sh were weighed and analysed
kg–1; D11 = 50 FTU · kg–1). The ingredient compositions for carcass composition. There was no mortality of fi sh
of the experimental diets are given in Table 3. A diet during the feeding trial. The water quality parameters
containing fi sh meal as the main protein source was used from each tank were monitored each week throughout
as the reference diet (RD). To each of the formulated the experimental period for temperature, pH, dissolved
diet 1% chromic oxide was added as digestibility oxygen, and total alkalinity. The ranges of water quality
marker. The diets were prepared in pelleted form using parameters were: temperature, 29–32°C; pH, 7–7.8;
0.5% carboxymethylcellulose as binder and the pellets dissolved oxygen, 4.6–5.5 mg · L–1; and alkalinity, 155–
were dried in oven and stored in airtight containers in a 170 mg · L–1.
refrigerator at 4ºC until used. The proximate composition Chemical analyses and data collection. Feed ingredients,
of experimental diets is presented in Table 4. experimental diets, faecal samples, and fi sh carcass
Experimental design. The feeding trial was conducted in (at the beginning and after the termination of experiment)
fl ow-through 90 L circular fi bre-glass tanks for 80 days were analysed for proximate composition according to
under laboratory conditions. Each tank was supplemented AOAC procedures (Anonymous 1990) and the procedures
with unchlorinated water from a deep tube well with as described by Roy et al. (2014).
continuous aeration. Rohu, Labeo rohita, fi ngerlings were Tannin and phytic acid contents in both fermented
obtained from a local fi sh seed dealer and acclimatized for and raw sesame oilseed meal were determined following
15 days and fed with a mixture of rice bran and mustard Schanderi (1970) and Wheeler and Ferrel (1971),
oil cake. The fi ngerlings (mean initial weight 1.85 ± 0.52 respectively. Phosphorus and calcium contents in the diets,
g) were randomly distributed in the fi bre-glass tanks at a faecal samples, and carcass were estimated following
stocking density of 15 fi sh per tank with three replicates Fiske and Subba Row as described by Hawk (1960) and
for each dietary treatment. Fish were fed once daily at Clark–Collip modifi cation of Kramer–Trisdall method
1000 h at a fi xed feeding rate of 3% of total body weight as described by Hawk (1960), respectively. Mn, Cu, and
per day. To determine the feed consumption, any leftover Fe contents were estimated using an atomic absorption
feed was collected 6 h after each feeding and weighed spectrophotometer (Electronics Corporation of India,
Table 3
Ingredient composition of experimental diets (% of dry matter basis)
Reference Sesame Diets with sesame seed meal Diets with sesame seed meal
diet diet fermented by LF1 strain fermented by Biophos-TS
Ingredient
RD D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
Fish meal 40.0 31.0 31.0 31.0 31.0 31.0 31.0 31.0 31.0 31.0 31.0 31.0
Mustard oil cake 24.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0
Rice bran 33.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0
Sesame seed meal — 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0
Soybean oil 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Cod liver oil 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Vitamin Premixa 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Chromic oxide 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Citric acidb — 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Dietary phytase
— — 10 20 30 40 50 10 20 30 40 50
[FTU · kg–1]
Sesame diet = diet with raw sesame seed meal; aVitamin and mineral mixture (Vitaminetes forte, Roche Products India Private Limited,
Mumbai, India); bCitric acid (0.5%) was added to the experimental diets for maintaining the pH at 5.5; Experimental diets and respective
phytase content [FTU · kg–1]: D2 = 10, D3 = 20, D4 = 30, D5 = 40; D6 = 50, D7 = 10, D8 = 20, D9 = 30, D10 = 40, D11 = 50; [1 FTU is
defi ned as the amount of phytase that liberates 1 μmol of inorganic phosphorus from 0.0051 mol · L–1 of sodium phytate per min at 37ºC
and pH 5.5].
Effi cacy of phytase in rohu nutrition 13
gaopufSa(wwtHTmdtwmohMonntrrtetheeyah ieaeooradafilfierrmti dtttewtilt Feeechn h yezgnrieir rrsu ioeontiasedemrio stthnnldiatddtenl ihqssaie ce ab so seroo r ueaoesatadap euarofne stoaffasltet(seditf fid el ue alnSptO E r i, h (wbcd tftnpa(etvAnflyfoieyeSA lceioaahfieg 1 fnr tNrGprrlainnfDcm pef yeic ohemoecaμdPlRashdsufnaey nyrih2rig U o n)gmgnnsstacb t0r au. cn[rrc mre)aoy 10eaSseo%aan ei f 9tne7fiawnettAwt ( Paii · t ca8)Act ’iaoonmdt.tsePrnael9a hciM .af Nar ssoHnt )emt ,o(sd(ye te i.rcnPOci1 fevAergd–ncue tawEam91istV aaezl ] rthnh9td(Rly,ntacyeAise A e,i4adpftar im f) w roec)nefi)silf,nmIP fe. e es faesanf opu a drOm l ehcwirsdyepnshn<n ar o.cndiieoseye eodne namois enrnmi0s dgs,gs gtn cpai mp.ipe iw 0tohvmeMgpho ouhM5 utLteanbposrse ya rossgi ieeairSsttsoatfi ndudadteatri 1i-nwf ocegioeodss E(9 ndannc elD fnn ex8f: p no,u ttee[ o ucc 5urhtedl%ln Alalobnenltn)reiylo utl.tg myeioc]A i rmtwr,eto aefws ose p(Srssnat oiin pU(oaatipnnfi4t Ffine rmrmn1betg.)1loi-Cgw 9td sci w2ewT rtl o5iaRettitaan9fihhhthanar5ir tyt))ydnceeeeeesss).,, Table 4(on dry matter basis) Diets with sesame seed meal fermented by commercially available phytase Biophos-TS D7D8D9D10D11 2 ± 2.6993.34 ± 2.7394.46 ± 2.8297.82 ± 2.8295.84 ± 2.764 ± 1.0335.00 ± 1.0135.24 ± 1.0235.37 ± 1.0235.14 ± 1.011 ± 0.248.87 ± 0.259.00 ± 0.269.19 ± 0.269.64 ± 0.287 ± 0.4415.64 ± 0.4515.75 ± 0.4515.87 ± 0.4515.92 ± 0.462 ± 0.185.94 ± 0.175.71 ± 0.165.47 ± 0.165.12 ± 0.158 ± 0.7827.89 ± 0.8028.76 ± 0.8331.92 ± 0.9230.02 ± 0.876 ± 0.1217.58 ± 0.1217.79 ± 0.1218.38 ± 0.1318.13 ± 0.13 7 ± 0.01NDNDNDND4 ± 0.01NDNDNDND –1ytase content [FTU · kg]: D2 = 10, D3 = 20, D4 = 30, D5 = 40; D6 = 50, –1us from 0.0051 mol · L of sodium phytate per min at 37ºC and pH 5.5];
Eccficrpp2fac0iFigaitoRnnnr eoooonarrr2.riseif5de cclEoorossmnmdhf.sas rr82uehettwiBe tcSdeeeegsm6%mccp 6eegaaeiiti,Untt%nndho. onssgupie et.1iooee h spLtrfirtinmp 5aFii fcddnrcehontttTcne %etaiobf s eiow aelardesorf.tSf aeafs nod mlriri etn2ocvll-goocftf hFm yrT5tro, efe inrmnme i m ineu.lnrnSiser6a. ar fi dp oehticn aan mT5Bv5aa(nceFfr enntt3%atla. ea itieraif e1rcbtloe0-iaonrenfirtlie5sn dleps poan-miatttfe F%ueolne eahrbiror y sdaT to enednumo5lraire ie nndf dUi s.mfttnlswo t Loat rre- sai Tiaff oTpoiiv neitFom· etBnttss6hmh nseiScnihe1k eooa.aeoeylamed 0 ssg nm B cnlt (at( a 0mdo a–ih ui34(cai1namle%siiTweot)0fl0ieellee inu,da ap ol ta e ,risicisd swb FFthanfin ze tshfloD BlilToTo ai ehaeen l tsfirctsifrdieeUicU9 ceticesoa-n1oh rdoLt reTh,el m peoa)n een dF(··ed.maSlhrtva c i ene1sTo sos nmakowkpaae,ni, feshl cugnfgh lta( tcueedo- r res3–t–ytalTriiaeoe ar11i a ng0uft )o)(ll lamhiScarn i .eTch dninso azuosna FvridteaieIlaem s en nn rpesTn bfi o aetal cdgolr l iinUlffsLi s0eeop bolnmor uger n2. Fotni oxs f22eddlhed·t5e imt1.tis7)r r inecce hm.s.uae fllk8% rrd aiee rTttlfiauugn4oaia mtrvtt no hhhedd–sg%rmattie1hddonnyeeeeeees)ll e composition of experimental diets based on sesame oilseed meal Diets with sesame seed meal fermented by LF1 strain D2D3D4D5D6 24 ± 2.6393.34 ± 2.6994.42 ± 2.7396.69 ± 2.7695.79 ± 2.6792.600 ± 1.0135.12 ± 1.0135.32 ± 1.0235.29 ± 1.0235.10 ± 1.0135.522 ± 0.248.64 ± 0.259.12 ± 0.269.37 ± 0.279.52 ± 0.278.432 ± 0.4415.08 ± 0.4315.47 ± 0.4515.64 ± 0.4515.75 ± 0.4215.124 ± 0.186.00 ± 0.175.50 ± 0.165.32 ± 0.155.30 ± 0.156.346 ± 0.7628.50 ± 0.8229.01 ± 0.8331.07 ± 0.8930.12 ± 0.8727.112 ± 0.1217.63 ± 0.1217.88 ± 0.1218.29 ± 0.1318.13 ± 0.1317.4 09 ± 0.01NDNDNDND0.011 ± 0.01NDNDNDND0.1 me diet =diet with raw sesame seed meal; Experimental diets and respective phs defi ned as the amount of phytase that liberates 1 μmol of inorganic phosphor
of 30 FTU · kg–1) and D5 (containing sesame oilseed meal mat 91.35.8.15.6.26.17. 0.0. SesaTU i
Rda(fssawooffsfPeorohpitniifogD erelrr oe dsa4m m n<itcw dte g 0aiahD iceeifih 0fi neptee od Fnht.6cddcpt 0 n tT aga gm eaD(5cgDgtnrUcadr )eooefrt9eoi 2nloow nduaon yn·.wtrwl ipd tir t f(k t(tfasa fnh%thhpPeg ehiteio neire–Lr c )otm1<f erir hfi)Fn nfapan .fi eos gt1 0rnwtFenhsoro .dsfm hoe0t aftr(tf ee iesro5Sesafl5dia dpsdm)enGnn0 i e wed n doccd euRc t Foeitaiiddhtif te) nitTfisfhii a,l tmaeeni vic Ut gpDz rtB ce f teenarg Doero5ioan·itsorrnrfii ltt1ioma o p ke cotcf fnwnhhigeetnsarndh enoor– tn (o 1 rmha tsmADet)garft l- f a rteyrTN1a lfiwioattni on0ihSvc(tuPoat edP,eiee (pU snea F F d s(gwDnt <no)CS C rwaco. ofe1 oG0RR y Hf4tici0u . g tRfi 0)0os orhwp h i(a 5nsw)Fw gctLaht,o)c Tionags e foewFdf nis Uvan e fifi1ih et(i e fldta n cPsi·rofrrsg i heaea, kdEwtn( h nrtr l%giinfiReiaeeetnoev–nloisst1))dygnnes)tttt,, Proxi Reference Sesame dietProximatedietCompositionRDD1 Dry matter [%]96.72 ± 2.7990.96 ± 2.63Crude protein [%]35.42 ± 1.0234.95 ± 1.01Crude lipid [%]8.07 ± 0.237.12 ± 0.21Ash [%]14.25 ± 0.4115.20 ± 0.44Crude fi bre [%]6.15 ± 0.189.72 ± 0.28NFE [%]32.83 ± 0.9523.97 ± 0.69Gross energy 18.21 ± 0.1316.83 ± 0.11–1[kJ · g]Tannin [%]ND0.29 ± 0.01Phytic acid [%]ND0.44 ± 0.01 Values are mean ± standard error of the mean (n = 3); D7 = 10, D8 = 20, D9 = 30, D10 = 40, D11 = 50; [1 FNFE = nitrogen-free extract; ND = Not detected.
14 Roy et al.
Apparent nutrient and mineral digestibility. The variables. Moreover, small fi sh are more sensitive to
apparent digestibility of protein, lipid, and ash were better diet differences; if small fi sh are unaffected, it is a safe
in fi sh fed diets D9 and D5, which were not signifi cantly assumption that larger fi sh will not be (Lovell 1998). The
(P < 0.05) different in the group of fi sh fed diets D6, results of our study indicated that the mean fi nal weight
D10, and D11. Maximum dry matter digestibility was of the fi sh increased considerably from the initial value
recorded in fi sh fed diet D2 and was not signifi cantly in all dietary treatments. The overall growth performance
different from that in the fi sh fed reference diet (Fig. 1). of rohu fi ngerlings, fed fermented sesame oilseed meal,
Maximum phosphorus, calcium, manganese, copper, and incorporated diets in both cases were better in comparison
iron digestibility was recorded in the group of fi sh fed diet to fi sh fed raw oilseed meal diet. The observation on
D9, which was not signifi cantly (P < 0.05) different in the weight gain in both cases i.e., phytase from fi sh gut
groups of fi sh fed diets D5, D6, and D10 (Figs. 2a and b). bacteria Bacillus licheniformis LF1 and commercially
Proximate carcass composition. Proximate carcass available phytase Biophos-TS corroborates the fi ndings
composition in experimental fi sh at the termination of Liebert and Portz (2005), who reported that different
of the feeding experiment is depicted in Fig. 3. The sources of microbial phytase similarly affected the growth
deposition of carcass protein and lipid was signifi cantly in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758).
(P < 0.05) maximal in fi sh fed fermented sesame seed The differences in the growth performance recorded in this
meal incorporated diets than the reference diet. The study agree with the report of Nwanna et al. (2005) that
crude protein, crude lipid, and ash contents of fi sh common carp, Cyprinus carpio Linnaeus, 1758, reared on
carcass were highest in fi sh fed diet D9, which was not phytase treated diets showed a signifi cantly higher weight
signifi cantly (P < 0.05) different from those in the group gain and feed conversion ratio in comparison to the diets
of fi sh fed diet D5. Mineral concentration in the carcass without phytase. Growth improvement was also observed
of experimental fi sh increased over the initial value in all in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)
dietary treatments and an increasing level of phytase was (see Yang et al. 2011); Nile tilapia (Hassaan et al. 2013);
associated with an increase in carcass mineral content zebrafi sh, Danio rerio (Hamilton, 1822) (see Liu et al.
(Table 6). The phosphorus, calcium, manganese, copper, 2013); Australian catfi sh, Tandanus tandanus (Mitchell,
and iron contents in the fi sh carcass were highest for diet 1838) (see Huynh and Nugegoda 2011); and yellow catfi sh,
D9, which were not signifi cantly (P < 0.05) different from Pelteobagrus fulvidraco (Richardson, 1846) (see Zhu et
those in the group of fi sh fed diet D5. In case of calcium, al. 2014) fed phytase supplemented diets. The dietary
no signifi cant (P < 0.05) difference was noticed in the fi sh microbial phytase pre-treatment improved growth in
fed diet D6 (containing sesame oilseed meal fermented fi ngerlings of Pangasius pangasius (Hamilton, 1822) (see
with bacterial strain LF1 at a concentration of 50 FTU · Debnath et al. 2005). In L. rohita also growth performance
kg–1) and D10 (containing sesame oilseed meal fermented was signifi cantly improved on corn gluten meal (30%)
with commercially Biophos-TS at a concentration of based experimental diets with graded levels of phytase
40 FTU · kg–1). pre-treatment (Hussain et al. 2011). However, phytase
Intestinal phytase activity. Intestinal phytase activity pre-treatment did not improve the growth of rainbow trout
in experimental fi sh at the termination of the feeding fed diets containing canola protein concentrate (Forster
experiment is presented in Fig. 4. The intestinal phytase et al. 1999), juvenile Korean rock fi sh, Sebastes schlegeli
activity increased with increasing level of incorporation Hilgendorf, 1880, fed diets containing soybean meal (Yoo
of bacterial or commercial phytase in the diets. Intestinal et al. 2005) and Atlantic salmon, Salmo salar Linnaeus,
phytase activity was higher in the groups of fi sh fed 1758, fed soy protein concentrate treated with graded
fermented oilseed meal incorporated diets in comparison levels of phytase (Carter and Sajjadi 2011). Nwanna and
to that fed raw seed meal incorporated diet. The highest Schwarz (2007) also found only marginal effects on the
phytase activity was recorded in the group of fi sh fed diet mean weight gain, specifi c growth rate, and feed conversion
D9 which was not signifi cantly different from that in the ratio when common carp was fed with phytase pre-treated
groups of fi sh fed experimental diets D5 and D11. diet. This discrepancy may be due to the differences in
Faecal phosphorus concentration. The faecal the phytase pretreatment methods of the feedstuff and
phosphorus concentration was signifi cantly higher in fi sh also different rearing conditions. Hauler and Carter (1997)
fed (Fig. 5) raw sesame seed meal incorporated diet than reported that phytase stimulated appetite which increased
the reference diet and an increasing level of phytase in the the growth of Atlantic salmon, S. salar. In channel catfi sh,
diets was associated with a decrease in faecal phosphorus Ictalurus punctatus (Rafi nesque, 1818), also phytase pre-
concentration. Maximum faecal phosphorus concentration treatment stimulated appetite and increased the growth
was detected in the fi sh fed diet D1, containing raw sesame through increased feed intake (Li and Robinson 1997).
oilseed meal. Formation of complex between sesame seed α-globulin
and sodium phytate has been described as a bi-phasic
DISCUSSION reaction (Rajendran and Prakash 1993). Initially phytase
In the presently reported investigation small fi ngerlings binds protein through strong electrostatic attractions
(mean initial weight 1.85 ± 0.52 g) of Labeo rohita were which is followed by slower protein–protein interactions
used to evaluate dietary effect on the growth of the fi sh. resulting in precipitation when the protein–phytate
Small fi sh respond faster than large fi sh to nutritional complex exceeds a critical size. It appears that phytase
Effi cacy of phytase in rohu nutrition 15
Table 5Growth performance and feed utilization effi ciencies in Labeo rohita fi ngerlings fed experimental diets, based on sesame oilseed meal, for 80 days Reference Sesame dietDiets with sesame seed meal fermented by LF1 strainDiets with sesame seed meal fermented by Biophos-TSdietParameter RDD1D2D3D4D5D6D7D8D9D10D11 aaaaaaaaaaaaInitial weight [g]1.85 ± 0.521.87 ± 0.521.85 ± 0.521.86 ± 0.521.84 ± 0.521.85 ± 0.521.85 ± 0.521.85 ± 0.521.85 ± 0.521.86 ± 0.521.84 ± 0.521.84 ± 0.52ccbbaaabbaaaFinal weight [g]3.88 ± 0.113.70 ± 0.114.06 ± 0.114.28 ± 0.124.51 ± 0.134.88 ± 0.144.72 ± 0.144.19 ± 0.124.44 ± 0.134.91 ± 0.144.72 ± 0.144.60 ± 0.13ddccbaacbaabWeight gain [%]110 ± 3.1798 ± 2.83119.75 ± 3.46130 ± 3.75145.23 ± 4.19160.85 ± 4.64154.92 ± 4.47127 ± 3.67139.82 ± 4.03164.25 ± 4.74157 ± 4.53150 ± 4.33aaabcdcbbddcFCR1.76 ± 0.051.80 ± 0.051.69 ± 0.041.55 ± 0.041.36 ± 0.041.14 ± 0.031.23 ± 0.031.60 ± 0.041.48 ± 0.041.06 ± 0.021.18 ± 0.031.29 ± 0.03SFeed intake 1.151.111.151.091.000.870.930.711.080.820.900.96ccbbaaabaaaaSGR0.92 ± 0.020.86 ± 0.020.99 ± 0.021.04 ± 0.021.11 ± 0.031.20 ± 0.031.17 ± 0.031.02 ± 0.021.09 ± 0.031.20 ± 0.031.16 ± 0.031.14 ± 0.03eeedcabadabcPER1.61 ± 0.041.59 ± 0.041.69 ± 0.041.84 ± 0.052.08 ± 0.052.48 ± 0.072.32 ± 0.072.76 ± 0.071.93 ± 0.052.68 ± 0.072.39 ± 0.072.20 ± 0.06decbbaacbbaaANPU41.26 ± 1.1928.85 ± 0.8344.51 ± 1.2846.84 ± 1.3649.34 ± 1.4357.67 ± 1.6658.00 ± 1.6745.75 ± 1.3248.84 ± 1.4150.09 ± 1.4458.26 ± 1.6855.76 ± 1.61 –1Values 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]: D2 = 10, D3 = 20, D4 = 30, D5 = 40; D6 = –150, D7 = 10, D8 = 20, D9 = 30, D10 = 40, D11 = 50; [1 FTU is defi ned as the amount of phytase that liberates 1 μmol of inorganic phosphorus from 0.0051 mol · L of sodium phytate per min at 37ºC and pH –15.5]; Feed intake was expressed in g · 100gBW of fi sh per day; FCR = feed conversion ratio; SGR = specifi c growth rate; PER = protein effi ciency ratio; ANPU = apparent net protein utilization; Mean values Swith same superscript letter in the same row are not signifi cantly different (P < 0.05); Statistical analysis was not possible as determination was performed on pooled samples. Table 6Mineral composition of the carcass of experimental fi sh (Labeo rohita fi ngerlings) at the start and end of the 80 day feeding experiment Diets with sesame oilseed mealMineral compositionInitialRDD1D2D3D4D5D6D7D8D9D10D11 efPhosphorus ± 0.6517.92 ± 0.5127.25d ± 0.78 30.86c ± 0.89 38.75b ± 1.12 44.63a ± 1.29 40.18b ± 1.16 26.96d ± 0.78 31.12c ± 0.90 45.08a ± 1.29 42.27a ± 1.22 40.81b ± 1.18 22.56g14.76 ± 0.42–1[mg · g(52.8)(21.4)(84.6)(109.0)(162.5)(202.3)(172.2)(82.6)(110.8)(205.4)(186.3)(176.4)]Calcium 82.64d ± 2.38 67.49e ± 1.95 138.46c ± 3.99 146.59b ± 4.23 154.47b ± 4.46 170.78a ± 4.93 166.73a ± 4.81 136.23c ± 3.93 150.29b ± 4.34 172.64a ± 4.98 166.32a ± 4.80 158.16b ± 4.56 f53.25 ± 1.53–1[mg · g(55.19)(26.74)(160.01)(175.2)(190.08)(220.71)(213.1)(155.8)(182.2)(224.2)(212.3)(197.0)]Manganese 10.47e ± 0.30 6.41f ± 0.18 12.64d ± 0.37 13.89c ± 0.40 16.04b ± 0.46 16.82a ± 0.48 15.60b ± 0.45 13.32d ± 0.38 14.76c ± 0.42 17.85a ± 0.51 16.34b ± 0.47 16.00b ± 0.46 g4.83 ± 0.14–1[μg · g(116.7)(32.7)(161.6)(187.5)(232.0)(248.2)(222.9)(175.7)(205.5)(269.5)(238.3)(231.2)]Copper 7.52c ± 0.21 3.98e ± 0.11 6.34d ± 0.18 7.86c ± 0.22 8.02c ± 0.23 9.87a ± 0.28 8.26c ± 0.24 6.44d ± 0.18 7.98c ± 0.23 10.00a ± 0.29 9.75a ± 0.28 9.09b ± 0.26 f1.91 ± 0.054–1[μg · g(293.7)(108.3)(231.9)(311.5)(319.8)(416.75)(332.4)(237.1)(317.8)(423.5)(410.4)(375.9)]Iron 34.69d ± 1.00 25.75e ± 0.74 34.64d ± 0.99 42.87c ± 1.23 45.92b ± 1.33 54.70a ± 1.58 47.40b ± 1.37 35.10d ± 1.01 43.37c ± 1.25 55.09a ± 1.59 50.00b ± 1.44 48.82b ± 1.41 e22.56 ± 0.65–1[μg · g(53.76)(14.1)(53.5)(90.0)(103.5)(142.4)(110.1)(55.5)(92.2)(144.1)(121.6)(116.4)] –1Values are mean ± standard error of the mean (n = 3); Experimental diets and respective phytase content [FTU · kg]: D2 = 10, D3 = 20, D4 = 30, D5 = 40; D6 = 50, D7 = 10, D8 = 20, D9 = 30, D10 = 40, D11 –1= 50; [1 FTU is defi ned as the amount of phytase that liberates 1 μmol of inorganic phosphorus from 0.0051 mol · L of sodium phytate per min at 37ºC and pH 5.5]; Mean values with same superscript letter in the same row are not signifi cantly different (P < 0.05); Values in parentheses indicate percent increase.
16 Roy et al.
120
Protein Lipid Ash
100 aA aA aA aA aA
%] 80 bB cC bB bB bA a* a* bB bBa* a* a* a*
y [ b* b* a*
bilit 60 c* c* c*
sti
e
g
Di 40
20
0
RD D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
EExxppeerriimmeentnatl aDli edtsiets
Fig. 1. Apparent nutrient digestibility of Labeo rohita fi ngerlings fed experimental diets for 80 days; Values are mean
± standard error of the mean; Error bars show deviation among three replicates; Means with different letters are
signifi cantly different (P < 0.05); Experimental diets and respective phytase content [FTU · kg–1]: D2 = 10, D3 = 20,
D4 = 30, D5 = 40; D6 = 50, D7 = 10, D8 = 20, D9 = 30, D10 = 40, D11 = 50; [1 FTU is defi ned as the amount of phytase
that liberates 1 μmol of inorganic phosphorus from 0.0051 mol · L–1 of sodium phytate per min at 37ºC and pH 5.5]
(both bacterial and commercial) in the presently reported with other hardy species such as tilapia and common carp
study partially prevented the formation of protein–phytate (Benkappa and Varghese 2003). Another possible reason
complexes by prior hydrolysis of phytate to release of poor fi sh growth might be due to low appetite and low
protein so that it becomes available for growth. In the feed utilization (Islam 2002).
presently reported, improvement in growth performance Dietary protein is the primary source of nitrogen,
of rohu fi ngerlings may be attributed to enhanced release which is the other enriching nutrient of major concern
of nutrients by breaking down the bonds between phytate– in aquaculture (Hardy 2010). In this experiment, an
protein complex. Improvement in growth performance of increase in body protein accretion and nutrient (crude
rohu fi ngerlings may also be attributed to the improved protein, lipid) digestibility was noticed in rohu fi ngerlings
use of phytate-P as well as phytate-bound protein. fed Biophos-TS pre-treated diet, D9. The performance
Feeding rate is important for the growth, feed conversion, of fi sh reared on the control and raw oilseed meal
nutrient retention effi ciency, and chemical composition incorporated diets were poor compared to fi sh reared
of fi sh (Hung and Lutes 1987). An optimum feeding on other experimental diets in terms of weight gain,
rate is helpful to minimize the feed loss, reduce water PER, FCR, deposition of crude protein and lipid in the
pollution, and decrease cost of aquaculture production carcass, and apparent nutrient (crude protein, lipid)
(Du et al. 2006). In earlier experiments in this laboratory, digestibility. However, the signifi cant increase in body
it was established that rohu fi ngerlings fed to apparent protein accretion and nutrient (crude protein, lipid)
satiation displayed a feed intake more uniform and close digestibility recorded from this study disagree with the
to 3% BW · day–1 fi xed feeding level (Bairagi et al. 2002, report of Yan et al. (2002), who demonstrated that weight
2004, Ramachandran and Ray, 2007, Roy et al. 2014). gain and dietary protein utilization of channel catfi sh were
Therefore, in the presently reported experiment also rohu not improved by phytase addition.
fi ngerlings were fed at a fi xed feeding rate of 3% BW · It has been shown that the addition of microbial phytase
d–1. In our study, the overall growth performance of rohu enhanced the availability of various minerals from the
fi ngerlings remained low compared to its growth in natural plant oilseed meals and improved their absorption. Mineral
and farm conditions. A similar growth trend has also been concentration of the carcass of experimental fi sh increased
reported with other nutritional experiments conducted over the initial value in all dietary treatments. Although the
on Indian major carps including rohu (Ravi and Devaraj deposition of phosphorus, manganese, copper, and iron in fi sh
1991, Bairagi et al. 2002, 2004, Benkappa and Varghese carcass was higher in rohu fi ngerlings fed Biophos-TS pre-
2003, Khan et al. 2012, Roy et al. 2014). As fi shes are treated diet D9, they were not signifi cantly different from
poikilotherms, drastic change in their surrounding water those in the fi sh fed bacterial phytase pretreated diet D5.
temperature will infl uence their metabolic processes, In the case of calcium deposition, there was no signifi cant
behaviour, migration, growth, reproduction, and survival difference among the fi sh fed diets D5, D6, D9, and D10.
(Fry 1971, Pörtner 2001). A possible explanation for this Biophos-TS pretreated diets showed satisfactory results
lower growth rate could be that Indian major carps are in terms of phosphorus-, calcium-, manganese-, copper-,
sensitive to environmental conditions and do not attain and iron digestibility which was not signifi cantly different
maximum growth in a confi ned environment compared from those in the fi sh fed bacterial phytase pretreated diet.
Effi cacy of phytase in rohu nutrition 17
A 70 Phosphorus Calcium
60 a
a a
a a
b
50 b
c A A c b A A A
%] d B
bility [ 40 D d C B C B
sti 30 D
e
g
Di 20
10
0
RD D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
ExpEexpriemrimeennttaall Ddiieetsts
B 120
Manganese Copper Iron
100 a a
b aa* a* aa* bb* a* aa* aa*
bility [%] 8600 dCd* de* cCc* Bb* A A A cCb* B A A B
sti D
e
g
Di 40
20
0
RD D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
ExpExepreirmimeennttaal lD dieitests
Fig. 2. Apparent digestibility of phosphorus and calcium (A) and manganese, copper, and iron (B) in Labeo rohita
fi ngerlings fed experimental diets for 80 days; Values are mean ± standard error of the mean; Error bars show deviation
among three replicates; Means with different letters are signifi cantly different (P < 0.05); Experimental diets and
respective phytase content [FTU · kg–1]: D2 = 10, D3 = 20, D4 = 30, D5 = 40; D6 = 50, D7 = 10, D8 = 20, D9 = 30,
D10 = 40, D11 = 50; [1 FTU is defi ned as the amount of phytase that liberates 1 μmol of inorganic phosphorus from
0.0051 mol · L–1 of sodium phytate per min at 37ºC and pH 5.5]
The phosphorus-, calcium-, manganese-, copper-, and iron salar (see Storebakken et al. 1998); striped bass, Morone
digestibility did not show any signifi cant difference among saxatilis (Walbaum, 1792) (see Papatryphon and Soares
the dietary groups D9, D10, D11, D5, and D6. The results 2001); European seabass, Dicentrarchus labrax (Linnaeus,
of the presently reported study revealed that fermentation of 1758) (see Oliva-Teles et al. 1998); common carp, Cyprinus
sesame oilseed meal by phytase from both Biophos-TS and carpio (see Nwanna and Schwarz 2007); Australian catfi sh,
Bacillus licheniformis LF1 signifi cantly increased available Tandanus tandanus (Huynh and Nugegoda 2011); and
mineral contents in feed, indicating that the indigestible juvenile olive fl ounder, Paralichthys olivaceus (Temminck
phytate-P and also other minerals (Ca, Mn, Fe, and Cu) et Schlegel, 1846) (see Yoo and Bai 2014). Apparent
might have been successfully converted to available form digestibility of phosphorus, calcium, manganese, iron, and
by phytase. Phytase from fi sh gut bacteria B. licheniformis zinc from barley, canola meal, wheat, and wheat middling
LF1 and commercially available phytase Biophos-TS, both by rainbow trout also has been reported to increase with
possess the ability to liberate bound phytate P, which resulted the addition of phytase (Gatlin and Li 2008). Omnivorous
in increased P and other mineral digestibility. Improvements fi sh species such as channel catfi sh, Ictalurus punctatus,
in utilization of dietary phosphorus from soybean meal has been shown to exhibit increased phosphorus utilization
based feedstuffs have been reported in various fi sh species, from soybean meal based diets with the supplementation of
such as rainbow trout, Oncorhynchus mykiss (see Sugiura phytase (Yan et al. 2002).
et al. 2001, Yang et al. 2011); Atlantic salmon, Salmo
18 Roy et al.
A
A
h100 B A
s B
A B B
d, 80 B B
pi
Li
e
ud 60 C C C
n, Cr a a b c
ei 40 d
ot D e
Pr f a* b* f a* a* a*
ude 20 ie* ge* he* gd c c* gc* c*
r
C
0
RD D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
ExpEexrpimeriemnetnatal l dDiieettss
Fig. 3. Proximate carcass composition (% wet weight; crude protein, crude lipid, ash) of Labeo rohita fi ngerlings at
the start and end of the 80 day feeding experiment; Values are mean ± standard error of the mean; Error bars show
deviation among three replicates; Means with different letters are signifi cantly different (P < 0.05); Experimental
diets and respective phytase content [FTU · kg–1]: D2 = 10, D3 = 20, D4 = 30, D5 = 40; D6 = 50, D7 = 10, D8 = 20,
D9 = 30, D10 = 40, D11 = 50; [1 FTU is defi ned as the amount of phytase that liberates 1 μmol of inorganic
phosphorus from 0.0051 mol · L–1 of sodium phytate per min at 37ºC and pH 5.5]
18
16 Phytase
a
a a
14 b
b
12
U]
vity [10 c
acti 8 d
me e e
y 6 f
z
n
E g
4
2
0 0
0

RD D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
ExEpxepreirmimeennttaall D dieitests
Fig. 4. Intestinal phytase activity of Labeo rohita fi ngerlings fed experimental diets for 80 days; Values are mean ±
standard error of the mean; Error bars show deviation among three replicates; Means with different letters are
signifi cantly different (P < 0.05); Experimental diets and respective phytase content [FTU · kg–1]: D2 = 10, D3 = 20,
D4 = 30, D5 = 40; D6 = 50, D7 = 10, D8 = 20, D9 = 30, D10 = 40, D11 = 50; [1 FTU is defi ned as the amount of
phytase that liberates 1 μmol of inorganic phosphorus from 0.0051 mol · L–1 of sodium phytate per min at 37ºC
and pH 5.5]
In the presently reported experiment, pH of the Vielma et al. 1999). Citric acid has been reported to
experimental diets was maintained at 5.5 by adding citric increase P bioavailability by dephosphorylation of phytate
acid. Several studies reported that microbial phytase has in vitro (Żyła et al. 1995). Baruah et al. (2007a, 2007b,)
two optimal peaks of activity, one at pH 5.0–5.5 and the and Sugiura et al. (2001) also reported that addition of
other at pH 2.5 (Simons et al. 1990). On the other hand, citric acid into the fi sh feed increased P utilization in Labeo
phytase activity changes along the digestive tract (Yi and rohita juveniles and also apparent absorption of Mg and P
Kornegay 1996). There are some evidences that pH of the in Oncorhynchus mykiss, respectively. In carnivorous fi sh
gastrointestinal tract affects the bioavailability of minerals like rainbow trout, production of acids assist in lowering
in fi sh (Sugiura et al. 1998, Vielma et al. 1999). In addition the dietary pH, but in agastric species like L. rohita, no
to their effect on intestinal pH, supplementary organic such mechanism exists. Hence, addition of organic acids
acids can also bind various cations along the intestine or acidifi ers may reduce the dietary pH, which in turn
(Ravindran and Kornegay 1993), resulting in increased may reduce the pH of the intestine and enhance mineral
intestinal absorption of minerals (Sugiura et al. 1998, absorption. Further, dietary acidifi cation may reduce