– Joaquin Percebal-Morlanes, DVM, MSc, MRCVS
Plants naturally store phosphorus in the seeds in form of phytate. This molecule (Fig 1) can be found in cereal grains, legumes and oilseeds (Ravindran et al., 1995) and form complexes in the seed with proteins; later on, the action of phytase enzymes breaks the molecule down making the phosphorus available for the development of the plant during germination (Carla E. Hegeman et al., 2001).
Feeds for animals based on cereals and legumes contain phytate of which approximately 28.2% is phosphorus (Ravindran V, 2010)
Phytate in feeds not only interferes with the absorption of phosphorus but this molecule can bind proteins (Selle et al., 2000), cations (Morris 1986) and form complexes with starch (Thompson and Yoon, 1984), lowering the availability of nutrients present in the diet for the chickens.
Plant-based feedstuffs must be formulated with more phosphorus available to the animal from inorganic sources such as mono- or dicalcium phosphate that is 95- 98% available to the broiler, adding cost to the production (Leeson S & Summers J, 2001). In addition to this problem, the undigested phosphorus in the phytic acid is excreted and when the manure is spread to agricultural land, it may lead to contamination of the land if the amount applied exceeds the amount of phosphorus taken by the crops and contamination of water due to high concentration of phosphorus being lixiviated (Pote et al., 1996).
Phosphorus is an important mineral for the nutrition of broilers as it is necessary for the formation and maintenance of bones, metabolism and fertility (in the case of broiler parent stock) and is a expensive feed component in the broiler diet.
There are different strategies in order to minimize the problems associated with the phytic acid present in the plant seeds and improve the utilisation of dietary protein, energy, phosphorus and other minerals by the broilers and reduce the excretion of phosphorus:
- Low phytate crops
- Addition of microbial phytase enzyme
- Control of Ca/tP ratio in the feed
- Addition of additives (vitamin D, citric acid)
Low Phytate Crops
There are mutations identified in some cultivars (maize, barley, rice) that have been selected for their low content of phytate in the seeds. Two low-phytate alleles have been identified (lpa1-1, lpa2-1); the content of phosphorus in these lines with the allele of low phytate content (lpa) is the same as the non-mutants lines but the amount of phytate-P is 50 – 95% lower, with more inorganic Phosphorous (Raboy V, et al., 2001).
Likewise, there are soybeans low-phytate variants that contain approximately 45-65% inorganic phosphorus whereas the non-mutants variants contain 10-15% inorganic phosphorus (Wilcox RJ et al., 2000).
Addition of Microbial Phytase Enzymes
Chickens have some specific and unspecific phytase activity mediated by enteric phytase and phosphatase enzymes respectively in the small intestine but this enteric phytase activity is insufficient to break down all the phytate ingested in a typical diet based on soya and corn. The addition of exogenous phytase enzyme helps to make more phytate-P available for the chickens and reduce the negative effects that phytate has on the utilization of minerals, proteins and energy (Cowieson AJ, et al., 2006).
Effect of exogenous phytase on protein and energy utilization
Phytate not only binds to dietary protein (Selle et al., 2000) and starch (Thompson and Yoon, 1984), making them less available for chickens but also interacts with enteric enzymes such as amylase (Knuckles BE & Betschart AA 1987) and proteases (Caldwell, 1992), provoking more secretion of enzymes, mucins and increasing losses of endogenous protein (Pirgozliev V et al., 2005).
In addition to that, phytate makes Na+ less available for the chicken in the lumen of the intestine, interfering with systems of transport of amino acids and glucose, Na+-dependant in the intestinal wall (Ravindran V, 2010). All these actions lead to a decrease in the digestibility of the dietary protein in the order of 9 – 14% (Knuckles et al., 1989).
Depending on the amount of phytate in the diet and phytase added, we may expect different digestibility and retention of amino acids (see table 1 & Fig 2), minerals and energy.
|Diets containing 5 g of casein and different amounts of phytate (IP6) and phytase|
|0.5 g IP6/0 FTU||0.5 g IP6/1000 FTU||1 g IP6/0 FTU||1 g IP6/1000 FTU|
Table 1 (Cowieson AJ, et al., 2006) DM: dry matter; TAA: total amino acid; TMN: true metabolizability of nitrogen
Digestibility of dry matter and utilization of the energy, amino acids, phytate-P and minerals increase when phytase is added to the diet (Fig 3)
There are different types of exogenous phytases commercially available from fungal (i.e..: Aspergillus niger, Aspergillus ficuum) to bacterial (i.e.: Escherichia coli) origin, being the phytases of bacterial origin more efficient releasing phosphorus form phytate (Augspurger RN , et al.2010).
The products commercially available, express the activity of the enzyme by phytase units (FTU). However, different phytases have different activities depending on pH (see Fig 4),
and the activity of the enzymes standardised at pH 5.5 may not be the best predictor of the biological activity (see Fig 5).
The enzyme activity measured at low pH (2.5 – 3.5) has better correlation with the biological performance in broilers than the phytase activity at 5.5 and in this case, increasing the particle size of the feedstuff of low-phosphorus diets with added phytase would keep the feed in the proventriculus and gizzard longer and release more phosphorus (Bedford M & Greiner R, 2010).
Despite the importance of the addition of microbial phytase for the improvement on the utilization of phosphorus in the diet, exogenous phytase hydrolyses less than 35% of dietary phytate in broiler feeds (Selle PH & Ravindran V, 2007). The use of low-phytate cultivars (high available phosphorus) along with exogenous phytase improves the performance of the flock, allowing a reduction of total phosphorus in the feed in the order of 23 – 28% and reduction of phosphorus excreted (Huff WE, et al.1998).
The future on phytase research is focused on including it at higher concentration in the feed, discovery of new phytase enzymes, combination of phytase with other enzymes (amylase, xylanase, …) and optimisation of Ca/P ratios (Ravindran V, 2010)
Ratio Ca/P in the Diet
High Ca/P ratios in the feed have a negative effect in the utilisation of phytate-P by the broilers (Ballam et al., 1984). It is thought that Ca binds with phytate, making the complex Ca-phytate less soluble and more resistant to the action of phytase enzymes. The deposition of minerals in the bone decreases with high Ca/P ration; the influence of Ca/P is greater when no exogenous phytase is added into the ration (see Fig 6).
Addition of Vitamin D3
Vitamin D3 improves the absorption of P in the intestine (Table 2) by activation of a transport system different to the transport of Ca in the intestine (Edwards, 1993) and it is been proposed that vitamin D3 also activates phytases and phosphatases to hydrolyse phytate (Biehl RR et al., 1995) with an additive effect when both vitamin D3 and exogenous phytase enzymes are incorporated in the feed (table 2 & Fig 7).
|Weight gain (g)||Tibia ash (g/100g)|
|Control + 20 mg vit D/kg||235||40.9|
|Control + 1200 FTU/kg||245||38.5|
|Control + 20 mg/kg + 1200 FTU/kg||253||42.7|
Table 2.(Biehl RR et al.,1995) Weight gain and tibia ash in chicks fed during 12 days with vit D or phytase or vit D+phytase added to the control diet
And as we saw with the addition of phytase, the addition of vitamin D3 also improves the metabolic energy of the diet (Table 3).
|Control diet||Control diet + 75 FTU||Control diet + 5mg vit D|
Table 3 (Edwards HM Jr, 1993)
Addition of Citric Acid in the Feed
It has been observed that supplementation of organic acids, especially citric acid has a positive effect in the utilization of phytate-P by the chickens (Boling SD, et al., 1998). Citric acid is also a potent chelator, so it would compete for Ca in the lumen of the intestine and that would reduce the number of phytate-Ca complexes, making phytate more vulnerable to hydrolysis (Boling SD, et al., 2000).
The effect of the addition of different amounts of citric acid to the diet can improve the weight gain (Fig 8) and the minerals deposited in the bones using diets deficient in phosphorus.
As mentioned before, citric acid binds Ca but does not interfere with the availability of Ca for the chickens in the intestine (Boling-Frankenbach et al., 2001).
Despite these positive effects on the utilization of phytate-P by the chickens with the addition of citric acid, the use of this organic acid in the diet at 4 – 6% is not economically viable, (Boling SD, et al., 2000), so it should be used in combination with the other measures already discussed. See Fig 9.
The addition of phytase in animal feeds may release phosphorus for the absorption in the intestine, increasing the energetic value of the feed and the availability of other nutrients such as amino acids and minerals. The cleavage of phytate makes more phosphorus available, improving the deposition of minerals in the bone and the growth of the flock, reducing the total amount of phosphorus needed in the feed and likewise reducing the excretion of phosphorus to the environment.
The action of this enzyme can be potentiated by the addition of vitamin D3 and organic acids.
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 One FTU is the amount of enzyme necessary to liberate 1mmol of iP/min from 0.00015 mol/L sodium phytate at ph 5.5 at 37oC (Ravindran V., et al, 2001)