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PHYTASE: AN EFFECTIVE TOOL TO HELP REDUCE FEED COSTS

Milan Hruby Ph.D. Technical Services Manager Danisco Animal Nutrition 8362 Tamarack Village, Suite 119-200 Woodbury, MN USA 55125
INTRODUCTION
Pig producers around the world face challenges in coping with increasing costs of production due to changes in ingredient feed pricing as well as trying to satisfy ever more stringent environmental standards. Feed enzymes will undoubtedly play an increasing and vital role in helping solve some of these challenges. For close to 15 years, feed phytase enzymes have clearly shown the opportunity to reduce feed costs and minimize waste production. New generation, bacterial origin phytases have shown superiority in liberating phosphorus (P) and calcium (Ca) from dietary phytate generating even greater economic benefits in their use. Additionally, new research studies have clearly shown the effect of phytase on improved digestibility of nutrients other than P and Ca. Furthermore, research focusing on various modes of phytase actions within the pigs gastrointestinal tract points to a more complex functioning of this enzyme. To fully embrace the greater potential of the new-generation phytase, poultry and pig producers can make greater reductions in dietary inorganic P in their diets (assuming that there is enough phytate P present), but also can reap the benefits of energy and amino acid reductions in the diet, due to the phytases effects on phytate as an anti-nutrient. What has become equally apparent with the recent rapid increase in phosphate and other feed ingredient prices is that customers have been interested in exploring uses of higher levels of phytase to replace more inorganic P especially in pig feeds with higher levels of phytate P. To calculate customer-specific matrix values for P and other nutrients according to the level of dietary phytate P, phytase dose together with some economic considerations, a commercially available software program based on a multi-factorial model - Phycheck - can be utilized by the pig industry providing another step in the optimization of phytase use.

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PHYTIC ACID AND PHYTASE

Pig producers face challenges of producing a consistently high quality meat cost effectively with a minimal impact on the environment. A very high proportion of P in cereal grains, oilseed meals and their by-products is bound within phytate (phytic acid, myo-inositol hexa-phosphate, IP6). Pigs lack the appropriate enzyme (phytase, Myo-inositol hexakisphosphate phosphohydrolase, Phytate phosphatase) to break down phytate, so a large proportion of P passes through the animal, thereby wasting this third most expensive nutrient and potentially causing environmental problems by polluting water sources. Furthermore, the polyanionic phytate molecule has a tremendous capacity to chelate cations and form various nutrient-phytate complexes resulting in not only a direct feed nutrient loss but also endogenous protein and energy losses. Phytase is present in nature and phytase activity was first detected in rice bran by Suzuki et al. (1907)! Since then phytase has been found intrinsically present in a number of feed ingredients including wheat (Peers, 1953) and barley; it is secreted by the small intestinal mucosa (Maenz and Classen, 1998 poultry) and can be found in the large intestine due to the action of microflora present there (Kerr et al., 2000). It is present in a majority of pig feeds today via addition of exogenous or feed phytase. While feed phytase was introduced commercially in The Netherlands in the early 1990s (Lenis and Jongbloed, 1999), the evidence of phytase effect on P and Ca digestibility has been known since the late 1960s (Nelson et al., 1968). The initial use of phytase was purely driven by environmental constraints related to P excretion. While this is still a very important reason for phytase use in pig feeds and will undoubtedly play an important role in the future (e.g Moratorium on pig production expansion in Manitoba, Canada), the effect of phytase on direct feed cost reduction is a driving factor behind todays widespread phytase use. Additionally, more efficacious second generation phytases further help increase an opportunity to use phytase even more economically in pig feeds.

DIFFERENCES AMONG PHYTASES ON THE MARKET

The first major phytases in commercial use were derived from different species of fungi (e.g. Aspargillus niger). Today, new generation phytases are derived from the bacterium E. coli. Data from studies conducted by a number of independent research centers and universities together with commercial experiences have shown that the new generation E.coli-origin (bacterial) phytases are more effective than traditional fungal phytases in pigs (e.g. Braa et al., 2006). The differences observed between these two types of phytases are probably best explained by differences in digestive effects of the enzymes on phytic acid. Indeed, there is some evidence that bacterial phytases have greater specificity for phytic acid

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(Wyss et al., 1999) compared to fungal phytases. Lim et al. (2000) reported that E. coli phytase, a member of the histidine acid phosphatase family, has the highest specific activity of all phytases characterized. Besides the difference in substrate specificity, the two types of phytase have different pH optima (Figure 1) and also Figure 1. pH optimum of fungal and bacterial phytases
100 % Relative activity in vitro 80 60 40 20 0 1.5 E. coli

2.5

3.5

4.5 pH

5.5

6.5

Fungal (3-phytase)

Fungal (6-phytase)
Reference: Danisco Animal Nutrition 2003

different affinity for the derivatives of phytate (because the rate of hydrolysis is reduced differently for phytate intermediates containing less phosphate groups) according to Lim et al. (2000). Table 1. The effect of pepsin on activity of various sources of phytase.
Origin of phytase [%] Residual phytase activity at pH 2.0 and 37C after 30 min of incubation

Pepsin ( 0 U)

Pepsin (1000 U)

Pepsin (3000 U)

A. niger P. Lycii E. coli

94.7 0.9 90.9 1.6 98.4 1.3

59.3 0.7 36.2 0.4 95.6 1.5

43.1 0.7 19.6 0.3 94.3 0.9

Greiner and Farouk (2007)

Results are the means of 4 determinationsstandard deviation

Additionally, considerable variation exists in the efficacy of phytase enzymes due to resistance to pepsin (Liu et al., 1998, Greiner and Farouk, 2007, Kumar et al., 2003; Table 1). According to Jendza et al. (2005) commensal bacteria like E. coli are capable of colonizing the gut of a pig because they have evolved mechanisms

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and enzymes well suited to compete and thrive in this environment, whereas fungal species such as Aspergillus niger are not found colonizing the gut because they lack the adaptations necessary to thrive in this particular environment. It is hypothesized that selecting a species of origin that is well suited to the pig gut will result in a highly efficacious enzyme. Besides substrate specificity, pH optima and resistance to breakdown by endogenous proteases, other characteristics such as temperature optima and possible differences regarding the effect of Ca:available P ratio on phytase activity are sometimes discussed when comparing two different sources of phytase (Braa et al., 2006). While the in vitro differences among two types of phytases are important to study and understand, the superior effect of bacterial-origin phytase on economics of pig production has to be clear to the final customer in order to make the use of bacterial-origin phytase over the fungal phytases relevant to the pig industry. Numerous research centers and universities across the world have compared bacterial-origin phytase with a fungal phytase in broiler, layer and pig feeds (Braa et al., 2006). The results of these trials demonstrated conclusively that the bacterial-origin phytase product is substantially more effective than the fungal phytase.

EXTRA-PHOSPHORIC EFFECTS OF PHYTASE

A number of research studies have concluded that phytase addition increases not only P and Ca digestibility but also protein, amino acid (Mroz et al., 1994; Biehl and Baker, 1996; Radcliffe et al., 2006) and energy digestibility together with increased plasma concentrations of glucose, insulin, urea N, and -amino acids (Johnston et al., 2004). While in poultry the energy digestibility responses are typically supported by a larger proportion of overall research studies, the effects are not as equally evident in studies focusing on digestible energy in pigs (Selle and Ravindran, 2008). It is suggested that determining the impact of phytase on net energy, rather than digestible energy in pigs would prove more instructive in studying phytase effects on energy improvement (Selle et al., 2008). Indeed, Zhang et al. (2006) reported that addition of bacteria-origin phytase into diets of 35-kg gilts subjected to an indirect calorimetry (using maintenance heat production and dietary heat increament to calculate net energy) significantly increased (P<0.05) dietary net energy. Regarding the mode of phytase action in terms of amino acid digestibility improvement, it is suggested that phytate depresses intestinal uptakes of amino acids, which is counteracted by phytase (Selle et al., 2007). Additionally, phytate has been shown to exacerbate endogenous amino acid flows at the ileal level in broilers (Cowieson and Ravindran, 2007; Cowieson et al., 2008). Phytateinduced increases in endogenous amino acid flows would stem from increased secretion and/or decreased re-absorption of amino acids (Nyachoti et al., 2000). On the energy side, the inhibition of alpha-amylase activity due to Ca-phytate interaction (Desphande and Cheryan, 1984) and a caloric effect of endogenous

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amino acids are typically reported as the reasons for energy effect of phytase rather than a direct complexing of starch with phytate (Thompson, 1988). At this point published research studies comparing bacterial and fungal phytases effects on amino acid and energy digestibility are limited. However, Cowieson et al. (2008) concluded that while the supplementation of both, bacterial and fungal, phytases in broiler feeds significantly reduced (P < 0.001) the flow of endogenous amino acids, the reduction tended (P = 0.06) to be greater for the bacterial phytase compared with the fungal phytase. These data suggest that the capacity of different phytases to counteract the anti-nutritive properties of phytic acid vary and that a substantial part of the amino acid and possibly energy responses observed following phytase supplementation, at least in broiler chickens, are due to reduced endogenous amino acid flows. Bacterial phytase (Phyzyme XP) has been compared against A. niger phytase (Natuphos) in digestibility and performance trials conducted in North and South America, Europe and Australasia contributing to a large number of individual data points. Performance responses indicated that the bacterial-origin phytase gave at least 20% higher performance improvements compared to the fungal-origin phytase (Figure 2). Furthermore, based on digestibility studies the nutrient improvements are recommended when the bacteria-origin product is used in commercial practice (Table 2).

Figure 2. The effect of two different phytases on pig gain

Bodyweight gain improvement (%)
10.00 8.00 % 6.00 4.00 2.00 0.00 0 100 200 300 400 500 600 700 800 900 1000 phytase (FTU/kg) Linear (Bacterial ) Linear (Fungal) Fugal-origin phytase y = 0.0062x P = 0.003 Bacterial-origin phytase y = 0.0091x

Bioefficacy ratio = 1.47:1 Bacterial-origin phytase is 1.47 times more effective than fungal phytase Bacterial phytase 25 treatment comparisons Fungal phytase 14 treatment comparisons Summary : 4 wheat & 3 corn-based trials All diets were reduced in calcium and phosphorus. Pig weight range: 8-112kg

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Table 2. Nutritive value contribution for bacterial-origin phytase (Phyzyme XP) in pig diets based on results from digestibility research studies at various phytase dosages.

Nutrient

Phyzyme XP Dose Rate (FTU/kg feed) 250 500 0.133 0.120 0.093 30.4398 0.1274 0.2444 0.0151 0.0046 0.0102 0.0237 0.0062 0.0404 750 0.163 0.147 0.114 32.4243 0.1357 0.2604 0.0161 0.0049 0.0108 0.0253 0.0066 0.0430 1000 0.184 0.166 0.129 33.8350 0.1416 0.2717 0.0168 0.0051 0.0113 0.0264 0.0068 0.0449

Total P (%)* Available P (%) Calcium (%) DE (kcal/kg) DE (MJ/kg) Digestible Protein (%) Digestible Lysine (%) Digestible Methionine (%) Digestible Cystine (%) Digestible Threonine (%) Digestible Tryptophan (%) Digestible Leucine (%)

0.082 0.073 0.057 27.0447 0.1132 0.2171 0.0134 0.0041 0.0090 0.0211 0.0055 0.0359

*Based on DCP (assumed availability 90%)

MULTI-FACTORIAL MODEL PHYCHECK

To calculate customer-specific matrix values for P, Ca, amino acids and energy according to the level of dietary phytate, phytase dose rate and economic considerations, customers can use a software program Phycheck - to obtain the highest benefit from the phytase product use. This service was developed based on the performance and digestibility research already mentioned in the text. The optimisation service employs a multi-factorial model to describe animal responses to phytase based on these trials. For example improvements in energy and amino acid digestibility according to phytase dose rate from 250 to 1000 FTU/kg feed. The model indicates improvements in dietary energy (ME, DE, NE), ileal protein and amino acid digestibility at any commercial feed phytate concentrations. An example of economic benefit using the matrix values predicted by the model for a Western Canada pig grower diet is presented in Table 3. Additionally, an economic comparison for 2 different enzyme doses with the dietary phytate in this ration at a sufficiently high level to evaluate this option more closely and a comparison with a fungal-origin phytase are presented.

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Table 3. Practical application of bacterial and fungal phytases in pig feeds
Ingredients Phytase Barley Wheat Corn SBM 48% Fat Limestone MDCP Salt Vit/Min Micro L-Lysine DL-Methionine L-Threonine Choline Chloride 60 Diet cost, $/MT Diff vs. no enz, $/MT 180 205 194 420 692 104 800 169 3100 2350 6700 3300 1570 $/MT - Enz NO 51.73 16 10 16.97 1.48 0.93 1.43 0.5 0.5 0.30 0.04 0.1 0.025 269 500 FTU/kg feed Natuphos 53.55 16 10 16.09 1.02 0.91 0.94 0.5 0.5 0.31 0.05 0.1 0.025 262.37 6.63 500 FTU/kg feed Phyzyme XP 54.27 16 10 15.87 0.57 0.98 0.84 0.5 0.5 0.31 0.04 0.08 0.025 257.57 11.43 1000 FTU/kg feed Phyzyme XP 54.86 16 10 15.67 0.38 1.00 0.61 0.5 0.5 0.31 0.05 0.08 0.025 255.32 13.68

Difference in cost as gross i.e. no enzyme cost included

SUMMARY AND CONCLUSIONS

While the economic justification for pig producers to use the new-generation phytase seems to be clear, there are still many areas of phytase and phytate research, which will require further focus. The effect of phytase on net energy, responses in low and high phytate feeds, Ca:avP ratio vs. phytase response and further research in the use of other feed enzyme activities together with phytase are just a few examples where the future of phytase research might lead us.

REFERENCES:
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Cowieson, A. J., V. Ravindran, and P. H. Selle, 2008. Influence of dietary phytic acid and source of microbial phytase on ileal endogenous amino acid flows in broiler chickens. Poult. Sci. 2008 87: 2287-2299. Desphande, S. S., and M. Cheryan, 1984. Effects of phytic acid divalent cations and their interactions on -amylase activity. J. Food Sci. 49, 516 519. Greiner, R., and A-E. Farouk, 2007. Purification and characterization of a bacterial phytase whose properties make it exceptionally useful as a feed supplement. The Protein Journal, 26, 7, 467 474. Jendza, J. A., R. N. Dilger, S. A. Adedokun, J. S. Sands, and O. Adeola, 2005. Escherichia coli phytase improves growth performance of starter, grower, and finisher pigs fed phosphorus-deficient diets. J. Anim. Sci. 2005 83: 1882-1889. Johnston, S. L., S. B. Williams, L. L. Southern, T. D. Bidner, L. D. Bunting, J. O. Matthews, and B. M. Olcott. Effect of phytase addition and dietary calcium and phosphorus levels on plasma metabolites and ileal and total-tract nutrient digestibility in pigs. J. Anim. Sci. 2004 82: 705-714. Kerr, M. J., H. L. Classen, and R. W. Newkirk, 2000. The effects of gastrointestinal tract microflora and dietary phytase on inositol hexaphosphate hydrolysis in the chicken. Poult. Sci. 79, (Suppl. 1) 11 (Abstr). Kumar, V., A. Miasnikov, J.S. Sands, and P.H. Simmins. 2003. Manipulating Pig Production IX. Editor J.E. Paterson. Proceedings of the Ninth Biennial Conference of the Australasian Pig Science Association, Fremantle, Western Australia, 23 26 November 2003, p 164. Lenis, N. P., and A. W. Jongbloed, 1999. New technology in low pollution swine diets: diet manipulation and use of synthetic amino acids, phytase and phase feeding for reduction of nitrogen and phosphorus excretion and ammonia emission Review Asian-Australasian Journal of Animal Science 12, 305 327. Lim, D., S. Golovan, C. W. Forsberg, and Z. Jia. 2000. Crystal structures of Escherichia coli phytase and its complex with phytate. Nat. Struct. Biol. 7:108113. Liu, J., D. W. Bollinger, D. R. Ledoux, and T. L. Venum. 1998. Lowering the dietary calcium to total phosphorus ratio increases phosphorus utilization in low-phosphorus corn-soybean meal diets supplemented with microbial phytase for growing-finishing pigs. J. Anim. Sci. 76:808813. Maenz, D. D., and H. L. Classen, 1998. Phytase activity in the small intestinal brush border membrane of the chicken. Poult. Sci. 77, 557 563.

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