phytase enzyme production

ABSTRACT

A technique was developed to produce crude microbial phytase (CMPhy) enzyme form Aspergillus niger, with the objective to enhance phytate phosphorus (pP) availability in corn and soybean meal based broiler diets. The results of in vitro experiments showed that CMPhy enzyme was most active at pH 2.0 and 5.0, as it results in the release of about 79.0% to 88.0% inorganic phosphorus (iP) as a percentage of total phosphorus (tP) from ground corn and soybean meal. In vitro pP hydrolysis was 87.14% in ground corn at 40°C and 5.0-5.5 pH after an incubation period of 8 hrs. with a ratio of 1:1 (CMPhy enzyme:substrate). The pP hydrolysis in soybean meal was 81.25% by applying the same conditions as for ground corn except an incubation period of 10 hrs. When CMPhy enzyme was added to low-P diets for broilers the availability of P increased (63.35%) as compared to the diet with normal-P contents (43.31%) supplied by dicalcium phosphate. Similarly P excretion was reduced to 20.04% units by CMPhy enzyme addition to low-P diet than normal-P diet. Microbial phytase addition to low-P diets, without affecting the performance reduced the P excretion in the dropping thus minimising the P pollution hazards.

Phytic acid can bound the free P and forms the compound phytate that reduces the availability of phosphorus (P) for monogastric animals. From two-thirds of the P in cereal grains and oil seed meals poultry can utilise only one-third of the P because they lack the phytase enzyme necessary to hydrolyse pP. Due to this reason protein, phosphorus and other minerals bound to phytic acid are excreted as such in the faeces of the birds. The excess of P and nitrogen excretion will not only causes a great threat to our environment but also results in an increase in feed cost due to supplementation of feeds with inorganic phosphorus salts.

The dire need to find an economical source of P for poultry rations prompted researchers to make available pP in the body of birds. This study was thus conducted with the objectives to produce phytase enzyme from a fungal species Aspergillus niger and to determine its efficacy for in vitro and in vivo dephosphorylation of corn and soybean meal

The enzyme phytase was produced from a fungus Aspergillus niger. Which was obtained from National Institute of Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. The phytase was produced through 10 days fermentation at 28°C on corn starch based medium containing (g/l): corn starch 91, glucose.1H₂O 38, KNO₃ 12.0, FeSO₄.7H₂O 0.20, KCl 0.60,  MgSO₄.7H₂O 0.60.  The pH was maintained at 4.5 by using 1N H₂SO₄, or 1N NaOH. After 10 days fermentation, the broth was subjected to a series of filtration by using cotton cloth and finally filter paper, until a clear filtrate was obtained. This filtrate in liquid form was then used as the crude phytase enzyme. The assay for phytase activity was carried out by using the method by Simons et al. (1990) and the amount of free phosphate was determined by method of Fiske and Subbarow (1925). One unit of phytase is the activity that liberates 1mmol phosphate from phytic acid in 1 min at pH 5.5 and 40°C. Activity of the enzyme was found to be 1.075  phytase units (PU) per min per ml of the crude culture filtrate at pH 5.5 and 40°C.

The in vitro enzyme efficacy was measured after the optimum conditions viz. enzyme:substrate ratio, incubation time, temperature and pH, were determined for the optimum release of free phosphate from ground corn (GC) and soybean meal (SBM). After determining these conditions the GC and SBM were treated with predetermined level of crude phytase enzyme for the degradation of pP. After drying the GC and SBM in the oven, they were used in three experimental broiler rations designated as control, N+Phyt and L+Phyt. Control and N+Phyt diets contained the untreated and treated GC and SBM, 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 GC and SBM, with the content of nP (0.36%), 20% less than the NRC (1994) recommendations.

The three experimental diets were fed to ninety day-old Hubbard broiler chicks randomly divided into nine experimental units of ten chicks each. Each diet was given to three experimental units of ten chicks each. Droppings collected per pen during the last three days of the fourth week (i.e., days 26, 27 and 28), were thoroughly mixed and weighed. Representative samples were dried in the oven at 60°C and ground to pass through a 1-mm sieve (Yi et al., 1996). These ground samples and feed 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). Birds per pen were weighed at weekly intervals and the data was analysed by using analysis of variance technique with completely randomised design. Treatment means were compared by using Duncan's multiple range test (Steel and Torrie, 1980).

RESULTS AND DISCUSSION

Enzyme phytase production was measured in terms of phytase activity, which was found to be 0.248, 0.279, 0.333, 0.384, 0.698, 0.885, 1.075 and 1.083, Phytase Units/ml/min. after 4, 5, 6, 7, 8, 9, 10, and 11 days of fermentation, respectively. A linear increase in phytase production was found with the days of fermentation. Maximum phytase activity (1.086) was obtained after 11 days of fermentation; however, the phytase activity was non-significant (P< 0.05) after 10, and 11^th day of fermentation Simons et al. (1990). Based on these observations, a time period of 10 days fermentation was used for the production of phytase enzyme.

                Phytase activity was found to be 1.075 Phytase Units/ml in 1 min at 40°C and pH 5.5, by using phytic acid as substrate. Results of the present study are in line with Shieh and Ware (1968), who reported phytase activity (mmoles P/min) of extracellular phytase produced by different strains of  A. niger (except soil isolates) in the crude culture filtrate from 0.268 to 1.129 PU/ml/min, at 37.5°C, using calcium phytate as substrate. However, the phytase activity is found to be lower as compared to the results Nelson et al. (1971), who reported an activity of 950 PU/gm in the acetone dried enzyme preparation from A.ficuum NRRL3135. This high phytase activity (Nelson et al., 1971) may be due to the use of different variety of Aspergillus for enzyme production. The most possible reason of high phytase activity is due to the physical nature of the enzyme, because enzyme preparation become concentrated as the crude culture filtrate get dried.

                The summary of conditions for optimum release of iP has been given in table 1. These optimised conditions were then applied for maximum release of iP from GC and SBM used in experimental rations and the results obtained have been give in table 2.

The results (table 2) showed that before phytase enzyme treatment, total phosphorus (tP), inorganic phosphorus (iP) and phytate phosphorus (pP) contents were 0.28, 0.08, 0.21 and 0.50, 0.20, 0.32% in GC and SBM, respectively. The degradation of pP after phytase enzyme treatment was 87.14 and 81.25% in GC and SBM, respectively. The results of optimised conditions and pP hydrolysis by phytase enzyme substantiate the findings of Han and Wilfred (1988) and Sandberg et al. (1996). Han and Wilfred (1988) reported that phytase from A. ficuum hydrolyzed about 85% pP in soybean meal at 50°C and pH 4-5.5. Sandberg et al. (1996) reported that phytase from A. niger showed pH optima at pH 2.0 and 6.0. They further reported that phytase activity occurred at all pH values between 1.0 and 7.5.

The effects of phytase supplementation on the performance of birds at the end of the experiment (28 days) are given in table 3. Compared to the control diet, the normal-P phytase (1.075 PU/gm) treated diet significantly (P< 0.05) increased the BW gain by 2.65%. The gain of the chicks fed on the low-P plus phytase diet was comparable to that obtained on the control diet (Table 3), which contained higher levels (1.1 vs 0.6%) of dicalcium phosphate (a source of inorganic P) to satisfy the birds requirement of non-phytate-P. Feed intake followed a similar pattern to that of BW gains (Table 3). 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 significantly  (P< 0.05) higher with he normal-P plus phytase diet at 28 days, than with the other two diets. It becomes evident that improved gains due to supplemental phytase were primarily because of the increased feed intake (Table 3). Thus dietary phytase supplementation did not affect the feed conversion ratio for either treatment. However, 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-significant. 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 3). Similar improvements in BW gain of broiler chicks with phytase supplementation of diets have been reported  by Simons et al. (1990). The improvement in growth performance of chicks fed on phytase treated diets may be attributed to the release of mineral from the phytate mineral complex and the utilisation of inositol by the bird (Simons et al. 1990) or increased starch digestibility. The same can also be due to the increased availability of proteins, because phytate also complexes with proteins, making them less available.

Average values of tP and Ca intake, outgo in excreta, and apparent availability, during the last three days (i.e., day 26-28) of the experiment have been shown in Table 4. Percent apparent availability of tP was higher(63.35 %) in phytase supplemented low-P diet and lower (43.31 %) 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. Total intake and outgo of Ca with the low-P plus phytase diet was significantly (P< 0.05) different from values for the 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 significant (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 findings are similar to those of many other workers (Simons et al., 1990  and Yi et al., 1996) who gave P-deficient maize and soybean meal diets to chicks. 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) 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. The reduction in P losses in the excreta can reduce the environmental pollution caused by P.

REFERENCES

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NRC. 1994. Nutrient requirements of poultry. National Research Council. 9^th Rev. ed. National Academy Press, Washington, DC.

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Shieh, T. R and J. H. Ware. 1968. Survey of microorganisms for the production of extracellular phytase. Appl. Microbiol. 16:1348.

Steel, R. G. D. and J. H. Torrie. 1980. Principles and Procedures of Statistics. 2^nd ed. McGraw Hill Book Co. Inc., New York.

Simons, P. C. M., H. A. J. Versteegh, A. W. Jongbloed, P. A. Kemme, P. Slump, K. D. Bos, M. G. E. Wolters, R. F. Beudeker and G. J. Verschoor. 1990. Improvement of phosphorus availability by microbial phytase in broilers and pigs. Brit. J. Nutr. 64:525-540.

Yi, Z., E.T. Kornegay and D. M. Denbow. 1996. Effect of microbial phytase on nitrogen and amino acid digestibility and nitrogen retention of turkey poults fed corn-soybean meal diets.  Poult. Sci. 75:979-990.

^abc Values within a classification in the same row followed by different letters are significantly different (P< 0.05).

^abc Values within a classification in the same row followed by different letters are significantly different (P< 0.05).