Phosphorus is a mineral that is essential to growth and development. Cereal grains and oil seeds contain substantial quantities of phosphorus, however up to 80% of the phosphorus is present as phytic acid. This poses a problem to monogastric animals because they do not produce sufficient amounts of intrinsic phytases necessary to hydrolyze the phytic acid complex. Because monogastric diets are mainly comprised of feedstuffs that have low phosphorus availabilities, phosphorus supplementation from inorganic sources is necessary in order to obtain optimal animal performance.
The low availability of phosphorus in plant ingredients poses problems both economically and environmentally. Economically, phosphorus is usually the third most expensive component in a monogastric diet after energy and protein. Environmentally, a large amount of consumed phosphorus is excreted in the feces and urine due to its high unavailability.
The beneficial effects of dietary supplementation of phytase were observed in layers, broilers, ducks, goose and pigs. Phytase supplementation in layers improved feed consumption, egg production and egg weights in birds fed low levels of available phosphorus compared to an unsupplemented treatment.
Until recently, phytic acid was considered primarily as a factor limiting phosphorus availability from plant- derived feedstuffs. Today, however, there is evidence that the deleterious effects of phytic acid go much beyond just limiting phosphorus availability. In its native stage, phytic acid is also complexed with various cations (Ca, Fe, Zn, Cu) protein and amino acids but also lipids and starch. Supplemental phytase has been shown to have the capacity to counter these anti-nutritional effects on protein and energy utilization.
Phytases are a group of enzymes that hydrolyze phytate to release phosphorus and other nutrients from the phytate complex. Phytases are widely found in different plant tissues or microorganisms. However, the activity of these phytases is highly variable and is influenced by several factors such as the pH value, temperature, moisture and substrate concentration or formulation technology used to stabilize the enzyme. In order to evaluate the efficacy of different phytase products, in vitro evaluations can give only an indication on the real effect of the enzyme in the intestinal tract of the animal. For a commercial evaluation of the value of a phytase product, the release of phosphorus and other nutrient has to be compared in vivo in the animal.
Phytic Acid: An Anti-nutritional Factor
In plant seeds about two-thirds of the phosphorus is stored as phytate (myo inositol hexakisphosphate). Phytates are binding mineral cations like Ca-Mg, K-Mg, Fe or Zn and form a poorly soluble complex. The main role of phytic acid in plant seeds is the storage of phosphorus, which is utilized during seed germination. Apart from minerals, phytic acid is also able to form complexes with proteins and amino acids. The amino group present on the side chain of amino acids is thought to be one of the main functional groups involved in protein-phytate interactions. Therefore, a significant proportion of amino acids that are frequently supplemented to poultry diets may complex with phytate. Such complexes decrease the digestibility of proteins and of supplemented amino acids. Figure 1 shows the possible interactions between phytic acid and different nutrients like phosphorus, calcium, trace elements and proteins.
The Use Of Phytase In Layer Diets
A correct determination of the efficacy of supplemented phytase in layer diets requires test diets, which are sufficiently low in phosphorus, so than an improvement in the phosphorus availability due to added phytase will be reflected in an improvement of performance criteria like laying percentage, egg weight, feed conversion ration or egg shell quality.
An extensive layer trial was conducted by Vahl et al. in 1993 to demonstrate the effectiveness or microbial phytase in diets low in available phosphorus compared to a supplementation with inorganic phosphorus. A basal diet containing an available phophorus content of only 1.8g/ kg feed was supplemented with incremental levels of inorganic phosphorus (MCP) resulting in 2.2, 2.6 or 3.6 g of available phosphorus per kg of feed. The diet containing only 1.8 g of available phosphorus was supplemented with 300 units of microbial phytase (Natuphos). Performance parameters like laying percentage, gram egg per day, feed per hen and day, as well as feed per egg were determined over a period of 21 to 40 weeks of age. The results of the experiment are shown in Figure 2. Performance of the negative control containing only 1.8 g of available phosphorus was set 100% and all changes in performance either as a result of inorganic phophorus addition or phytase supplementation were expressed relative to the negative control. Laying percentage, gram egg per day as well as kg feed per kg egg were improved with addition of inorganic phosphorus to the negative control proving that the phosphorus level in the negative control was deficient for the laying birds. Available phosphorus levels higher than 2.6 g per kg of feed did not further improve layer performance. The supplementation of the diet deficient in available phosphorus with 300 units of phytase improved performance to a level even higher than the highest addition of inorganic phosphorus proving the 300 units of phytase can easily compensate for 0.8 available phosphorus. This conclusion can be made as the added phytase compensated for the difference between the negative control (1.8 g available phosphorus) and the treatment containing 2.6 g available phosphorus. A further increase in the available phosphorus level to 3.6 g per kg feed could not improve performance. Therefore other nutrients than only (other than) phosphorus are responsible for the additional performance effects in the phytase treatment.
Phytase is not only known to release phosphorus from the phytate complex, but also releases amino acids, protein and energy. Using digestibility measurements, these effects have been quantitied for phytase (valid for Natuphos only) and a matrix value sytem has been developed. This matrix value system allows to include phytase into least cost formulation. A layer experiment has been conducted at the University of the Philippines Los Banos in 2000 to evaluate the effect of microbial phytase in layers using the matrix value system for feed formulation. A commerial layer diet was used as the reference group. This commercial diet was reformulated using the matix value system developed for microbial phytase (Natuphos). The reformulated diet was supplemented with 300 phytase units. Table 1 shows the diet composition, the nutrient contents as well as the diets cost. Feed cost using the matrix values and microbial phytase were reduced significantly by US$6.
Performance was monitored from week 24 to week 35 and the results are summarized in Table 2. The results of this practical experiment show that phytase can improve the utilization of other nutrients besides phosphorus. The supplementation of a diet formulated with the matrix values developed for phytase (Natuphos only) kept performance at the same level as in a commercial layer feed, and at the same time, reducing feed cost by US$6.
The Use Of Phytase In Duck Diets
The effectiveness of microbial phytase in laying hens has been demonstrated in a large number of experiments whereas the number of trial with ducks is less. The following example shows that phytase is a very effective tool in ducks too.
A total of 1200 Chinese Shaoxin ducks were housed in floor pens from 26 weeks of age up to 36 weeks of age. The dietary treatments included a positive control with a nutrient content representing a practical commercial situation and a negative control with a reduced specification for total phosphorus from 0.65% (positive control) down to 0.45% (negative control). This phosphorus reduced diet was supplemented with incremental levels of phytase (300, 400 or 500 units/kg diet). The diets consisted mainly of corn, soybean meal, wheat bran and fishmeal. Details of the diet composition as well as the nutrient contents are shown in Table 3.
To evaluate the effects of a reduction of inorganic phosphorus and supplementation with microbial phytase, the parameters feed intake, egg production percentage, egg weight and feed conversion efficacy were determined. All data are summarized in Table 4. Neither reduction of phosphorus content of the diets nor the addition of microbial phytase effected feed intake of the laying ducks during the 10 week trial. Egg weight was not influenced by the treatments, with a tendency of lower egg weights in the group containing the low phosphorus content supplemented with 500 ulits/kg diet.
Egg production in the positive control group was 78.5% and was reduced to 74.5% in the negative control with lower phosphorus content. This indicates that the phosphorus supply was below the requirement of the ducks at this stage of production. Addition of phytase significantly increased egg production compared to the negative control at all dosages and also compared to the positive control group at addition rates of 400 units and 500 units/kg. Notice that the highest level of phytase supplementation (500 units) resulted in egg production which was 11 and 15 percentage points higher than the positive and negative control treatments respectively.
Feed conversion in the positive control was 3.49 kg/kg . Reduction of phosphorus content (negative control) tended to impair feed conversion ratio (3.58). Addition of 300 units of phytase/kg diet improved feed conversion compared to the negative control (3.39 vs.3.58). Compared to the positive control the addition of 400 units tended to improve feed conversion and 500 units resulted in a significant improvement in feed conversion, to 3.16.
The economic impact of replacing inorganic phosphorus (DCP) by supplementation with microbial phytase was evaluated by comparing costs of the commercial duck diet (positive control) with the diet reduced in DCP and supplemented with 500 units of phytase/kg. The treatment containing 500 units/kg was selected for this comparison because this level of phytase has shown the greatest beneficial effects with respect to laying performance and feed conversion ratio. Reduction of phosphorus content in the phytase supplemented group resulted in a replacement of 13 kg of DCP by only 100 g of microbial phytase. Other changes in the feed formula to make up the free space in the diet as a result of the replacement of 13 kg of DCP by 100 g of phytase had a further impact on the cost of the different feed formulas. The cost evaluation for all diet ingredients as influenced by the inclusion of phytase are shown in Table 5.
Adding extra corn, wheat bran and rapeseed meal to the diet adds cost whereas the reduced inclusion level of soybean meal saves costs. Reducing DCP affected not only the phosphorus level but also the calcium level of the diet. Therefore additional limestone has been added back to the diet to reach a calcium level which meets the requirement of the ducks. Overall this calculation shows that reduction of phosphorus level and supplementation with 500 units of phytase resulted in net cost savings of 2.6 USD per tonne feed in comparison with a commercial laying duck diet. Calculations, which take the higher egg production of the 500 units of phytase treatment into account, would result in even greater profits for phytase use.
The Efficiency Of Different Phytase Sources
Intensive research tests have shown that the fungus Aspergillus ficuum produces the highest phytase activity compare to other fungi. Phytase from Aspergillus ficuum is a so called 3-phytase as it starts its initial hydrolysis of phytate on the 3 position of the phytate ring. Recently a new phytase product has been introduced to the market, which is a phytase from Peniphora lycii. This phytase starts the hydrolization of the phytate ring on position number 6 instead of position 3. Consequently it can be assumed that the properties and the mode of action of both enzymes are quite different. In order to evaluate the efficacy of different phytase products in vitro evaluations can give only an indication on the real effect of the enzyme in the intestinal tract of the animal.
Besides the temperature optimum, the pH profile of an enzymes is the most important parameter to evaluate the efficiency of an enzyme under certain conditions. Figure 3 shows the relative phytase activity of two phytases. Pig or broiler feed normally has a pH value of around 6. In the stomach, the pH is reduced to a value between 2 and 3 by the secretion of HCl. At a low pH of about 3 Aspergillus ficcuum phytase has a 20 percentage points (0.6 vs.0.4) higher relative activity than Peniphora lycii phytase. At a higher pH of 6, which is relevant for the small intestine of pigs and the crop of poultry, Aspergillus ficcum again shows a higher activity than Peniphora lycii phytase.
Although the parameter described above is of interest to characterize phytases, finally phytase has to work in the animal¡ãOs digestive tract and has to be bio-effective in releasing nutrients. In the cas of phytase, phosphorus is the target nutrient. The bio-efficacy of phytase compared to a standard phosphorus source can be measured in an animal trial measuring performance or bone parameters. A basic diet deficient in phosphorus is supplemented with graded amounts of monocalcium phosphate to establish a dose response curve and to calculate the response per unit of monocalcium phosphate added (see Figure 4). Likewise, the basal phosphorus deficient diet is supplemented with graded levels of the phytase sources (see Figure 5). A dose response curve can than be determined and the release of phosphorus compared to monocalcium phosphate can be calculated. A scientifically unacceptable approach to compare the efficacy of different phytases is to take a positive control with an adequate phosphorus level, reduce this phosphorus level by a set amount and measure the response on the addition of different phytases. This trial design can not determine the true release of phosphorus, as the adequacy of the phosphorus level relative to the needs of the animals is unknown. If diets are only marginally deficient in phosphorus the release of small amounts of phosphorus can restore the phosphorus adequacy and therefore differences in the efficacy of different products never will be detected.
In order to determine the bio-efficacy of the two phytase sources, a phosphorus deficient feed was used as a control diet. This phosphorus deficient diet (0.20 % of available phosphorus) was either supplemented with different amounts of phosphorus from MCP (0.2, 0.4 or 0.6 g phosphorus addition) or with increasing levels of each phytase (100, 200 or 300 phytase units/kg). Figure 4 shows the effect of increasing supplementation of inorganic phosphorus from MCP on weight gain of broilers. The regression equations shows that weight gain increased by 141.79 g with the addition of 1 g of available phosphorus from monocalcium phosphate. The correlation between weight gain and phosphorus addition was very close with a correlation coefficient of more than 99%.
In Figure 5, the effect of the supplementation with the three levels phytase from Aspergillus ficuum and Peniphora lycii on weight gain is shown together with the regression equations and the correlation coefficients. The best response in weight gain on phytase addition could be measured for Aspergillu ficuum phytase. Weight gain increased by 0.3247 g per phytase unit. When Peniphora lycii phytase was added to the phosphorus deficient basal diet, weight gain increased only by 0.1470 g per phytase unit.
The comparison of the response curves for the Aspergillus ficuum and Peniphora lycii supplementation show that more than double of the Peniphora lycii phytase has to be used to achieve the same weight gain as with Aspergillus ficuum phytase.
Phytase has been studied extensively in laying hens, breeders and ducks to allow reliable conclusions about the release of phosphorus from phytic acid. It has been calculated from long term production trials as well as from direct measurements in the gastrointestinal tract that 300 units of phytase (only valid for Natuphos phytase from BASF) are equivalent to 1 g of phosphorus from MCP or 1.14 g of phosphorus from DCP. This replacement of inorganic phosphorus has a significant impact on the feed and production cost. The effects of phytase on the utilization of protein and energy are furthercontributing to the economical benefit of using phytase. These effects of microbial phytase on the utilization of protein, amino acids and energy have been quantified and summarized in a matrix value system. Using this matrix value system phytase can be used as a feed ingredient in least cost formulation reflecting the release of nutrients from the phytic acid complex. Practical performance trials have shown that microbial phytase is a very efficient tool to improve the profitability of feed and animal production as documented in the above described layer and duck trials.
For a complete evaluation of the value of phytase not only the effects on animal performance and feed cost but also the efficacy of different products have to be taken into consideration.
With respect to the efficiency of different phyases, the data presented above clearly prove that phytase derived from Aspergillus ficuum is more effective in hydrolizing phytic acid than phytase derived from Peniphora lycii. Broiler tests using formulated dry phytase products demonstrated that phytase produced with Aspergillus ficuum is 100% more effective than phytase produced with Peniphora lycii.
Vahl, H.A., G.J. Borggreve and H.P. Stappers, 1993. The effect of microbial phytase in layer feed. CLO-Schothorst experimental report No 374 (NL).