Effect of microbial phytase produced from a fungus
Aspergillus niger
on bioavailability of phosphorus
and calcium in broiler chickens
Tanveer Ahmad
*, Shahid Rasool, Muhammad Sarwar,
Ahsan-ul Haq, Zia-ul Hasan
Department of Animal Nutrition, University of Agriculture, Faisalabad, Pakistan
Received 24 February 1999; received in revised form 12 August 1999; accepted 18 October 1999
Abstract
Phytase was produced from a fungusAspergillus nigerthrough 10-day fermentation in a maize starch-based medium. Activity of the enzyme was found to be 1.075 phytase units per minute per ml of the crude culture ®ltrate, at pH 5.5 and 408C. A 4-week feeding trial was conducted on 90-day-old broiler chickens, fed on diets based on maize and soyabean meal, to study the ef®cacy of phytase enzyme on growth performance, apparent availability of P and Ca, tibia-and-toe ash and mineral contents of tibia. Treatments involved a normal-P level (control, 4.5 g/kg non-phytate phosphorus, nP), a normal-P diet with phytase enzyme-treated maize and soyabean meal and a low-P diet plus phytase (1.075 PU/gm of substrate). Phytase supplementation increased (p< 0.05) BW in normal-P plus phytase diet by 2.65 percentage units as compared to control. Gain in weight was almost similar with both, low-P plus phytase, and control diets (645 vs. 653 g). Added phytase increased the feed intake in the same manner as weight gain, however, feed conversion ratio (FCR) was similar to that for all diets. Phytase treatment of low-P diet increased (p< 0.05) the relative retention of P and Ca by 20.1 and 5.0 percentage units, respectively, than with the control diet. The tibial-and-toe ash contents were increased (p< 0.05) by the normal-P plus phytase diet than with the control diet. Tibial-and-toe ash contents of low-P plus phytase diet were comparable (51.2 vs. 48.6 and 10.9 vs. 10.5) with that of the control diet. Phytase treatment had no effect on the concentration of any of the minerals measured in whole tibia ash, but did increase (p< 0.05) P and Ca in tibia DM of chicken by 4.5 and 9.8 percentage units, respectively, in normal-P plus phytase, and 1.2 and 2.2 percentage units, respectively, in low-P plus phytase diets. These results show that microbial phytase treatment of a low-P diet increased growth and relative retention of P and Ca, and improved bone mineralization in broiler chickens.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Phytase; Phosphorus; Calcium;Aspergillus niger; Bioavailability; Broiler 83 (2000) 103±114
*Corresponding author.
1. Introduction
The availability of phosphorus (P) in plant sources is limited by the presence of a naturally occurring compound, phytate. Salts of phytic acid (phytate) contain about two-thirds of the P in cereal grains and oil seed meals, the major components of poultry feed. Phosphorus and other minerals when bound to the phytic acid become poorly available, as shown in man, chickens and pigs (Ketaren et al., 1993; Kornegay and Qian, 1994; Broz et al., 1994; Anonymous, 1996). Insoluble protein phytate complexes are formed below the isoelectric pH of proteins, reducing Ca, Mg, Fe, and Zn absorption from the intestinal tracts of animals and man (Reddy et al., 1982).
Since the birds lack the phytase necessary to hydrolyse phytates they can utilise only one-third of the P of plant origin. The protein, P and certain other minerals bound in phytic acid are excreted in the faeces and, thus, these have to be supplemented in greater amounts. The cost of the traditional inorganic P supplements for poultry diets (dicalcium phosphate, DCP) has also increased considerably. Hence, improvements in the availability of phytate phosphorus (pP) are needed.
Supplementation of feeds with phytase (myoinositol hexaphosphate phosphohydrolase) that cleaves the ortho-phosphate groups from the phytate molecule (Gibson and Ullah, 1990), enables monogastric animals to absorb P, bound in the phytic acid-chelate com-plex, thus decreasing the need for inorganic P supplements. Phytase supplementation of broiler diets improved growth and bone mineralization, and decreased mortality (Qian et al., 1996). Dietary P requirement can be decreased by 1 g/kg by phytase supplementa-tion of the diet (650±900 units/g feed), without in¯uencing the performance of the birds (Vogt, 1992). In broilers P excretion can be decreased up to 50% and an immediate reduction of 30% appears practically possible (Schoner, 1992).
The objective of this study was to synthesize microbial phytase enzyme by using a fungus Aspergillus niger and to determine its effect on dietary P and calcium (Ca) bioavailability and performance of broiler chickens, fed on diets based on maize and soyabean meal.
2. Materials and methods
2.1. Enzyme production
The phytase was produced from a fungus Aspergillus niger through 10-day fermentation at 288C on maize starch-based medium.1 After 10-day fermentation, the broth was subjected to a series of ®ltrations by using cotton cloth and, ®nally, ®lter paper, until a clear ®ltrate was obtained. This ®ltrate in liquid form was then used as the crude phytase enzyme. The ®ltrate was stored at 48C for not more than 7 days.
1Containing (g/l): maize starch 91, glucose. 1H
2O 38, KNO312.0, FeSO47H2O 0.20, KCl 0.60,
MgSO47H2O 0.60. The pH was maintained at 4.5 by using 1 N H2SO4, or 1 N NaOH.
2.2. Assay of enzyme
The ®ltrate obtained was assayed for its phytase activity by using the method of Simons et al. (1990). The assay was carried out at 408C in a shaking water bath. The reaction was initiated by mixing 0.1 ml of ®ltrate and 0.9 ml of 0.1 M sodium acetate buffer adjusted to pH 5.5 and containing phytic acid at a ®nal concentration of 1 mg/ml. A time course of 60 min was taken by terminating the reaction after 15, 30 and 60 min by adding 6 M HCl to a ®nal concentration of 1 M. Samples to which HCl had been added before the addition of phytic acid served as a blank for the contents of free phosphate. After termination of the reaction, the samples were centrifuged for 10 min at 3000 rpm. The amount of free phosphate was determined spectrophotometrically by the method of Fiske and Subbarow (1925) in the clear, supernatant fraction. One unit of phytase is the activity that liberates 1mmol phosphate from phytic acid in 1 min at pH 5.5 and 408C. The activity of the enzyme was found to be 1.075 phytase units (PU) per min per ml of the crude culture ®ltrate at pH 5.5 and 408C.
2.3. Enzyme treatment of substrate
The conditions, viz. enzyme : substrate ratio (1 : 1), incubation time of 8 h for corn and 10 h for soyabean meal at 5.5 pH and 408C were determined for the optimum release of free phosphate. The maize and soyabean meal were treated by spraying with a predetermined level of crude phytase enzyme for the enzymatic degradation of pP. After enzyme treatment, these ingredients were dried at 608C in the oven for about 12 h and used in broiler rations (Table 1).
2.4. Experimental diets
Three isocaloric and isonitrogenous diets were prepared (Table 1) containing treated, and untreated, maize and soyabean meal. These diets were designated as control (with normal P contents), NPhyt (normal-P plus phytase) and LPhyt (low-P plus phyatse). Control and NPhyt diets contained the untreated, and treated, maize and soyabean meal, respectively, and were prepared according to the National Research Council (NRC, 1994) requirement for available P (non-phytate P, nP). Low-P plus phytase diet was prepared to contain the enzyme treated maize and soyabean meal, with a content of nP (3.6 g/kg), 20% less than the NRC (1994) recommendations. Ground limestone was added to each diet to maintain calcium : total phosphorus (Ca : tP) ratios of 2 : 1 as recommended by NRC (1994).
2.5. Experimental birds
experimental units of 10 chickens each. Feed and water were offered ad libitum during the entire experimental period.
2.6. Collection of excreta
At the end of the third week wire mesh of 2.5 cm size was placed in each pen at a height of about 15 cm from the ¯oor. Plastic trays were placed under the wire mesh during the last 3 days of the fourth week (i.e. days 26, 27 and 28), for the total collection of excreta. Excreta were collected separately from each pen. During the collection period, birds were fed ad libitum. At the end of the collection period, excreta of each pen were thoroughly mixed and weighed. Representative samples of the excreta were dried in an
Table 1
Composition of experimental diets (g/kg)
Ingredients Control NPhyta LPhytb
Maize 350 350 350
Soyabean meal 190 190 190
Rice 192 192 192
Cottonseed meal 23 23 28
Corn gluten meal (660 g crude protein/kg) 70 70 70
Fish meal 80 80 80
Vitamin and mineral premixc 5 5 5
Calculated analysis
aNormalphytase (diet containing phytase-treated maize and soyabean meal with normal level of P). bLowphytase (diet containing phytase-treated maize and soyabean meal with low level of P). cSupplied per kg of diet: vitamin A, 1500 IU; vitamin D
3, 200 ICU; vitamin E, 10 IU; vitamin K, 0.5 mg;
thiamine, 1.8 mg; ribo¯avin, 3.6 mg; pyridoxine, 3.0 mg; vitamin B12, 0.009 mg; pantothenic acid, 10 mg; niacin, 27 mg; choline, 500 mg; biotin, 0.15 mg; folic acid, 0.55 mg; m[TA1]anganese, 60 mg; zinc, 40 mg; copper, 8 mg; iron, 80 mg; and antioxidant (Santoquin(r)), 125 mg.
dAfter addition of enzyme treated of maize and soyabean.
oven at 608C and ground to pass through a 1-mm sieve (Yi et al., 1996b). These ground excreta samples were used for the analysis of P (AOAC, 1984) and Ca (Richard, 1954). All calculations were expressed on a DM basis.
Feed consumption by the birds was recorded on a pen basis at weekly intervals and daily for the last 3 days of week 4 (i.e. days 26±28). Samples of each diet were analyzed for DM, total P (AOAC, 1984) and Ca (Richard, 1954). Birds per pen were weighed at weekly intervals. The average BW, on a pen basis, was thus used for the statistical evaluation of treatments. Mortality, if any, was also recorded.
2.7. Sampling and measurement of tibia and toes
Tibia-and-toe samples of one bird (selected at random) from each experimental unit (3 birds/diet) were collected after slaughtering the birds at the end of Week 4. Left tibia (with cartilage caps) of each killed bird was excised and cleaned of adhering tissues. Tibia samples were dried to a constant weight at 1008C in an oven, and then ashed at 6008C for 4 h in a muf¯e furnace for the determination of bone ash, which was expressed on dry weight of the tibia (Scheideler, 1993 ). The ash from the tibia was solubilized with a nitric and perchloric acid mixture (5 : 3, vol/vol) in 100 ml conical ¯asks, and the volume was made to 100 ml with distilled water (Yi et al., 1996a). Determination of P and Ca contents was made from this sample as in the samples from feed and faeces.
Toe samples of the killed birds were obtained by severing the middle toe through the joint between the second and third tarsal bones. The left and right middle toes of one bird within a pen were obtained, yielding two samples of toes per pen (these were averaged for the statistical evaluation of treatments). The clipped toes were cleaned of any waste material, but were left intact otherwise. No ¯esh, skin, or toe nail was removed. The toe samples were dried to a constant weight at 1008C and then ashed in a muf¯e furnace at 6008C for 4 h (Potter et al., 1995). Toe ash was expressed as a percentage of dry weight.
2.8. Statistical analysis
The data on performance, apparent availability of P and Ca, tibia-and-toe ash percentages and weight, tibial mineral contents were subjected to statistical analysis for interpretation of results by using analysis of variance technique with completely randomized design (CRD). Treatment means were compared by using the Duncan multiple range test (Steel and Torrie, 1980).
3. Results and discussion
3.1. Birds performance
Table 2
The effect of phytase supplementation on feed intake, weight gain, and feed conversion ratio of broiler chickens fed on diets containing enzyme-treated maize and soyabean meal
Parameters 0±14 days 0±21 daysa 0±28 daysa
Control NPhyt LPhyt SE Control NPhyt LPhyt SE Control NPhyt LPhyt SE Feed intake (g) 230 231 228 0.9 725 c 751 a 701 b 7.7 1183 b 1198 a 1137 a 9.6 Weight gain (g) 216 219 211 2.4 540 b 573 a 536 b 7.4 869 b 892 a 856 b 6.5 Feed : gain 1.06 1.05 1.08 0.012 1.34 1.31 1.31 0.014 1.36 1.34 1.33 0.014
aValues within a classi®cation in the same row followed by different letters are signi®cantly different (p< 0.05).
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age. However, these effects were signi®cant (p< 0.05) at 21 and 28 days of age. Compared to the control diet, the normal-P phytase (1.075 PU/gm) treated diet consistently increased the BW gain, however, the improvement was signi®cant (p< 0.05) only at 21 and 28 days, when phytase treatment increased the gain in chickens by 6.11 and 2.65%, respectively.
The gain of the chickens fed on the low-P plus phytase diet, at 14 days of age, was comparable to that obtained on the control diet (Table 2), which contained highs (11 vs. 6 g/kg) of dicalcium phosphate (a source of inorganic P) to satisfy the birds requirement of nP. From 14 to 28 days of age, the increase in BW with control and low-P plus phytase diets was almost equal (i.e. 653 and 645 g, respectively).
Increase in BW was more with the normal-P plus phytase diet than with the too low-P plus phytase diet (Table 2) by about 6.92 and 4.23% at 21 and 28 days of age, respectively.
Total feed intake followed a pattern similar to that of BW gains (Table 2). Added phytase in both normal-P and low-P diets increased the feed intake, yet it was even higher (by 5.37%) with the normal-P diet. Feed intake was found to be signi®cantly (p< 0.05) higher with he normal-P plus phytase diet, both at 21 and 28 days, than with the other two diets.
The improved gains due to supplemental phytase were primarily because of the increased feed intake (Table 2). Thus, dietary phytase supplementation did not affect the feed conversion ratio for either treatment. However, at 28 days FCR was better with the normal-P plus phytase (1.34) and low-P plus phytase (1.33) diets than with the control diet (1.36), yet the differences were statistically non-signi®cant.
The results revealed that supplementation of broiler diets with phytase at 1.075 PU/g of feed can reduce the tP and nP requirements by 15 and 20%, respectively, from the value recommend by NRC (1994), without having any adverse effect on BW gain (Table 2).
Similar improvements in BW gain of broiler chickens with phytase supplementation of diets have been reported by Simons et al. (1990) and Broz et al. (1994). The improvement in growth performance of chickens fed on phytase treated diets may be attributed to the release of mineral from the phytate mineral complex and the utilization of inositol by the bird (Simons et al., 1990) or increased starch digestibility (Knuckles and Betschsrt, 1987) The same can also be due to the increased availability of proteins, because phytate also complexes with proteins, making them less available. Phytate-protein complexes are less subject to proteolytic digestion than the same Phytate-protein alone. So it can be postulated that phytase-liberated proteins from the complex, making them more available to the birds.
weeks of age with supplemental phytase. A lower requirement of P at 4 weeks of age than at 2 weeks of age and an adaptation of the birds to utilize more pP in critical circumstances might explain these ®ndings.
3.2. Apparent availability of phosphorus and calcium
Average values of tP and Ca intake, outgo in excreta, and apparent availability, during the last 3 days (i.e. days 26±28) of the experiment are presented in Table 3. The effect of phytase treatment on tP retention was signi®cant (p< 0.05). The maximum (1.6 g) tP intake during days 26±28 was for the normal-P plus phytase diet, while the minimum (1.3 g) was for the low-P plus phytase diet. The maximum tP outgo in faeces occurred with the control diet (0.9 g) as compared to the above-mentioned respective diets (0.5 and 0.8 g). Apparent availability of tP was higher (63.4 g/100 g) for the low-P plus phytase diet and lower (43.3 g/100 g) in control diet.
In the case of Ca, total intake was higher for the normal-P plus phytase diet than for the control and low-P plus phytase diet. On the other hand, the outgo of Ca (as tP outgo) was higher for the control diet than for the other diets. Total intake and outgo of Ca with the low-P plus phytase diet was signi®cantly (p< 0.05) different from values for the control and normal-P plus phytase diets. Percent excretion of Ca was also lower in low-P plus phytase diet as compared to control and normal-P plus phytase diets. Apparent availability of Ca was higher in phytase-treated low-P diet and lower in control diet; however, these differences were not signi®cant (p> 0.05).
As expected, phytase treatment of the low-P diet increased (p< 0.05) the P retention by 20.04 and 12.73 percentage units as compared to control and normal-P plus phytase diets, respectively (Table 3). These ®ndings are similar to those of many other workers (Simons et al., 1990; Broz et al., 1994; Schoner et al., 1993; Yi et al., 1996a, b), who gave P-de®cient maize and soyabean meal diets to chickens. Simons et al. (1990) indicated that addition of phytase to low-P diets increases the availability of P to over 60 g/100 g and the amount of P in the droppings decreased by 50 g/100 g, when compared to a low-P diet without enzyme supplementation.
Phosphorus excretion on the low-P diet decreased (p< 0.05) with the addition of phytase and this might have increased the availability of both, P and Ca (Table 3),
Table 3
Effect of phytase treatment on apparent availability of total phosphorus and calcium in broiler chickens fed on diets containing enzyme-treated maize and soyabean meal from days 26 to 28
Parameters Total phosphorusa Calciuma
Control NPhyt LPhyt SE Control NPhyt LPhyt SE Intake (g) 1.6 a 1.6 a 1.3 b 0.14 3.1 3.2 2.7 b 0.13 Excreta output (g) 0.9 a 0.8 a 0.5 b 0.13 1.9 1.8 a 1.5 0.12 Apparent availability
(g/100 g)
43.3 a 50.6 ab 63.4 a 3.45 39.8 42.7 44.8 1.81
aValues within a classi®cation in the same row followed by different letters are signi®cantly different
(p< 0.05).
because both are part of the same complex and are released by the phytase enzyme at the same time. The increase in the availability of both, P and Ca decreases the amount of P in the droppings owing to a better balance of the two minerals. Many other researchers (Simons et al., 1990; Schoner et al., 1991; Schoner, 1992; Kwon et al., 1995) have also reported a reduction of P excretion from 30 to 60 g/100 g. The reduction in P losses in the excreta can reduce the environmental pollution caused by P.
Phytase treatment of low-P diets increased (p< 0.05) the Ca retention by 5.0 and 2.1 percentage units as compared to control and phytase-treated normal-P diets (Table 3). This improvement was expected because phytase liberates Ca from the Ca-phytate complex and as the availability of P increases, the availability of Ca also increases, because both are part of the same complex. The retention of both P and Ca was higher in phytase-treated normal-P diet as compared to the control diet, but it was signi®cantly (p< 0.05) lower when compared with phytase-treated low-P diet (Table 3). This may be due to the fact that control diet contained normal levels of P and Ca, which satisfy the birds requirements, whereas phytase treatment of this diet increased both P and Ca contents which were not fully retained by the bird and led to excessive amounts being excreted. On the other hand, with the low-P diet which was less by about 20% in nP, the phytase treatment increased the P and Ca contents in such amounts that were ef®ciently utilized by the birds. That is why retention of both the minerals was higher with the low-P plus phytase diet. Improvements in the utilization of Ca by supplemental phytase have been reported by Ketaren et al. (1993), Mitchel and Edwards (1996), and Zyla et al. (1996).
3.3. Tibia-and-toe DM and ash contents
The effect of phytase supplementation on the tibial bone-and-toe DM and ash contents as well as tibial bone Ca and P contents of 28-day-old broilers are shown in Table 4. Phytase treatment of diets improved both, the DM and ash contents of the tibia and toe.
Table 4
Effect of phytase treatment on the tibial bone and toe dry matter, ash and tibial bone contents of total phosphorus and ash in broiler chickens fed on diets containing enzyme-treated maize and soyabean meal
Parameters Tibial contentsa Toe contentsa
Control NPhyt LPhyt SE Control NPhyt LPhyt SE Dry matter (g) 4.0 4.4 4.1 0.19 2.1 2.3 2.2 0.08 Ash (g/100 g) 48.6 b 62.0 a 51.2 b 4.12 10.5 b 12.1 a 10.9 0.39 P (g/100 g of DM) 7.6 b 12.1 a 8.8 b 0.87
P (g/100 g of Ash) 15.4 16.5 17.2 0.58 Ca (g/100 g DM) 17.1 b 26.9 a 19.3 b 1.81 Ca (g/100 g Ash) 34.8 36.8 37.5 0.92
aValues within a classi®cation in the same row followed by different letters are signi®cantly different
The highest total dry weights (4.3 and 2.3 g) of tibial bone and toes were recorded in birds fed on the normal-P plus phytase diet followed by those fed on the low-P plus phytase diet and control. However, the differences among the three diets were statistically non-signi®cant (p> 0.05). Total ash contents of the tibia and toe were signi®cantly (p< 0.05) higher (62.0 and 12.1 g/100 g, respectively) with the normal-P plus phytase diet. The tibial-and-toe ash concentrations of the birds fed on control and low-P plus phytase diets were not signi®cantly (p> 0.05) different.
3.4. Tibia P and Ca contents
The effects of phytase supplementation of diets on Ca and P content of tibial bone DM of the birds were signi®cant (p< 0.05) (Table 4). Phytase treatment of low-P diet increased the P and Ca content in the tibia DM by 1.2 and 2.2 g/100 g, respectively, as compared to control diet. The increase in P and Ca (4.5 and 9.8 g/100 g) was more pronounced (p< 0.05) in phytase treated normal-P diet. The concentrations of the two minerals in the ash of tibia were relatively constant and were not much affected by phytase supplementation (Table 4).
Phytase treatment of diets has been reported to increase the tibia ash content by many workers (Nelson et al., 1971; Perney et al., 1993; Broz et al., 1994; Sebastian et al., 1996). It is considered to be a good indication of increased bone mineralization, associated with phytase supplementation and consequent increased P and Ca availability. As the availability of P increased, the availability of Ca also increased and both were deposited in the bones (Simons et al., 1990). Phosphorus and Ca together account for >50% of the bone ash content (Qian et al., 1996) and any treatments which results in their increased bioavailability will have positive effects on bone DM and ash contents. None of the treatment affected the concentration of P and Ca in whole tibia ash, but phytase supplementation signi®cantly improved the content of P and Ca in the DM of tibia. Broz et al. (1994) revealed that phytase supplementation of a maize and soyabean meal diet increased the tibia ash of broiler chickens.
4. Conclusion
The DM and ash concentrations of tibia and toe were directly proportional to the P and Ca bioavailability and closely related to the weight gain of the birds. The BW gain, percentage tibia-and-toe DM and ash of chickens fed on diets containing 4.7 g/kg nP was comparable to that of chickens fed on a diet containing 20% less nP, but supplemented with phytase, indicating that phytase supplementation increased the pP utilization in the low-P diet.
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