Maximising the response to phytase

Accumulation of in the environment due to the disposal of animal waste has forced the livestock industry to better use the phosphorus available in feed ingredients. Phytase has proved able to release this phosphorus if it is used in the right way. What

NUTRITION has to be done to maximise its response?

By Dr. Thau Kiong Chung, Roche Vitamins Asia Pacific Pte Ltd, Singapore

he disposal of animal waste leading to the accumulation of phosphorus in Tsoils and subsequently its entry into surface and ground waters has sparked off When using phytase to improve P utilisation, don’t forget that other major environmental concerns. Poultry ingredients can affect the ’s efficacy. (Photo: World Poultry) feeds contain ingredients of plant origin from which 50 – 80% of the total phospho- Table 1. The content of total, phytate, nonphytate, digestible and available phosphorus and rus is present as phytate phosphorus. The digestibility and bioavailability of phosphorus in selected feed ingredients for poultry inability of poultry to utilise phytate phos- phorus, due to lack of endogenous phytase, results in a substantial excretion of phos- Ingredient tP1, pP1,npP2, Digestibility3, dP, Bioavailability4,aP, phorus into the environment. In the ab- %%%%%%% sence of phytase, inorganic feed phos- Corn 0.24 0.17 0.07 29 0.07 19 0.05 phates are often added to diets to meet the Wheat 0.27 0.19 0.08 38 0.10 46 0.12 phosphorus needs of poultry. Rice bran 1.31 1.05 0.26 16 0.21 25 0.32 Continued pressure to reduce phospho- Wheat bran 0.99 0.79 0.20 37 0.37 29 0.29 rus excretion has instigated the search for Rapeseed meal 0.70 0.41 0.29 33 0.23 59 0.41 alternatives that may help to improve di- Soybean meal 0.39 0.23 0.16 42 0.16 39 0.16 etary phosphorus utilisation. Phytase of mi- Meat& meal 5.00 0.00 5.00 61 3.05 81 4.05 crobial origin is becoming more affordable Fish meal 2.20 0.00 2.20 74 1.63 100 2.20 and economical for use in feed applica- MCP 22.00 0.00 22.00 85 18.70 100 22.00 tions, so it is one of the alternatives that DCP, hydrated 18.00 0.00 18.00 80 14.40 100 18.00 help to reduce phosphorus excretion by DCP, anhydrous 18.00 0.00 18.00 70 12.60 95 17.10 poultry by at least 30 to 40%. In order to ful- tP = total phosphorus; pP = phytate phosphorus; npP = nonphytate phosphorus; dP = digestible phosphorus; aP = available ly explore the potential of phytase, it is of phosphorus paramount importance to identify factors 1 Data (mean values) adapted from published literature and inorganic feed phosphate manufacturer’s specification that potentially influence its responses in 2 npP = tP – pP practical diets. 3 Data adapted from CVB (2000) and De Bruyne and Von Felde, 2000. 4 Data adapted from Cromwell and Coffey, 1993 and Axe, 1998; relative to the availability of phosphorus in monosodium Phosphorus Terminology phosphate, which is assuming to be 100%. There are several terms being used to ex- press the phosphorus requirement as well as the availability of phosphorus in feed in- rus availability is assumed to be 100%. losses are negligible when the animal is fed gredients and inorganic feed phosphates. Available phosphorus values are relative below its phosphorus requirement. Terms such as available phosphorus, di- and comparative, and therefore they are not Therefore, apparent digestibility values are gestible phosphorus and nonphytate phos- absolute values. often used in practice. phorus are often used erroneously and in- Digestible phosphorus is defined as that Nonphytate phosphorus is a chemically terchangeably. Moreover, they refer to very part of the phosphorus in the feed that is defined entity, albeit it cannot be chemical- different “types” of phosphorus, which are not excreted when the animal is fed below ly analysed. It is derived mathematically by intimately related to the types of methods its phosphorus requirement. Standardized subtracting analysed phytate phosphorus used to evaluate them. methods for determining digestible phos- from analysed total phosphorus. Available phosphorus (relative bioavail- phorus (Van Der Klis and Versteegh, 1996; Table 1 shows the different types of phos- ability) is often derived from linear and Eeckhout and De Paepe, 1997) are used for phorus from feed ingredients and inorganic curvilinear regression slope-ratio growth such endeavours. Apparent phosphorus di- feed phosphates. and bone assays (Chung and Baker, 1992; gestibility values are without correcting for Sullivan et al., 1992; Eeckhout and De endogenous losses, whereas true phospho- Feed formulation Paepe, 1997). It is defined as the phospho- rus digestibility values take into account of Practical poultry diets devoid of animal rus availability of a test source relative to a endogenous contribution. It is also as- protein meals usually contain 0.25 – 0.40% reference source from which the phospho- sumed that the endogenous phosphorus phytate phosphorus. There are adequate

WORLD POULTRY - Elsevier Volume 18,No 8.'02 www.AgriWorld.nl 17 substrates present in these diets for phytase Table 2. Bioefficacy of supplemental phytase at differing dietary levels and calcium: to work on. The response to phytase may be phosphorus ratios on growth performance and mineral retention in 0 – to 21-day-old broiler much greater in diets containing high levels chickens fed low-phosphorus, corn-soybean meal diets of dietary phytate phosphorus or . When formulating diets for a specific type of phosphorus, be it total, non-phy- Dietary Calcium, % 0.60 0.60 1.00 1.00 1.25 1.25 tate, digestible or available (relative), one Dietary total phosphorus, % 0.54 0.54 0.54 0.54 0.54 0.54 must recognise the importance of matching Ca: total phosphorus ratio 1.11 : 1 1.11 : 1 1.85 : 1 1.85 : 1 2.31 : 1 2.31 : 1 the dietary phosphorus content con- Phytase, U/kg diet 0 600 0 600 0 600 tributed by feed ingredients and inorganic feed phosphates to the phosphorus require- Weight gain, g/21 day 591 616 555 636 530 577 ment of the bird. Feed intake, g/21 day 780 811 783 830 701 771 Feed:gain, g/g 1.45 1.40 1.55 1.40 1.45 1.45 Dietary P levels P retention, % of intake 54.7 57.6 57.5 61.2 53.2 59.0 The dietary level of phosphorus influences Tibia ash, % DM 25.1 26.5 26.2 29.5 24.0 28.8 the animal’s response to added phytase. Source: Sebastian et al., 1996 The response to phytase is much greater in diets containing lower levels of non-phy- tate, digestible or available phosphorus, ganic phosphate. Differences in dose-rate occur, they may undermine the bioefficacy NUTRITION compared to those containing levels near or recommendation observed between these of phytase and the growth and bone perfor- at the requirement of the animal. Similarly, two phytases arise from due to differences mance of broiler chickens as illustrated by the response to added phytase may be in enzyme origin, sequence, Sebastian et al. (1996) in Table 2. The nega- higher at high dietary levels of phytate physical and biochemical properties, pH tive effect of imbalanced dietary calcium to phosphorus. This is relevant to the typical profile and reaction kinetics. Thus, it is ob- phosphorus ratios on growth performance feed formulations used in Asian countries vious and to be expected that the activity is more dramatic without phytase supple- where dietary phytate phosphorus content dose rates of Peniophora phytase are differ- mentation under the conditions of excess is generally high. ent from those of Aspergillus phytase. calcium and deficient phosphorus. Addition Phytase, just like other , re- of phytase improves growth performance as Phytase product form sponds linearly initially and curvilinearly a result of improved dietary calcium: phos- When selecting phytases for commercial subsequently to graded levels of phytase ac- phorus ratios and increased levels of phos- applications, it is very important to have tivity. In other words, there is a diminishing phorus liberated from phytate by phytase. some in-depth knowledge about the prod- response to phytase addition after reaching Imbalances of dietary calcium: phospho- uct form, as this is associated with its stabil- the threshold or optimum level. rus ratios are usually due to calcium excess- ity during storage and feed Manufacturers of phytase usually recom- es in practice. Excess calcium precipitates and processing conditions. Commercial mend the optimal dose phytase unit, which phytate, forming insoluble calcium-phytate phytases are available as solid or liquid is experimentally determined to be the complexes that are resistant to phytase hy- forms. The solid form can be either pow- most economical and cost effective for drolysis. On the other hand, excess calcium dered or granulated. Commercially, the commercial applications. increases the pH of intestinal content and granulated form is formulated by advanced subsequently decreases phytase activity formulation technologies, which produce Phosphorus equivalency value and reduces solubility and absorption of phytase products differing in heat toler- Phosphorus equivalency value is a term used minerals. Moreover, excess calcium sup- ance. The liquid form of the phytase is rec- to describe the replacement or substitution presses phytase activity by competing for ommended for aggressive and harsh pro- value of phytase. It is defined as the amount the active site of the enzyme. For practical cessing of feeds. The liquid enzyme spray- of inorganic phosphorus that can be re- purposes, a dietary calcium: total phospho- ing system is best installed before the load- moved from the diet by a given phytase unit rus ratio of 1.2 (1.1 – 1.4): 1 is recommend- out and after the siever. The ultimate objec- of activity. For direct comparison of equiva- ed. While a dietary ratio of 2.2 (2.0 – 2.4): 1 tive of selecting the most appropriate prod- lency value of phytase for phosphorus and is suggested for calcium: non-phytate, di- uct form is to ensure the delivery of stable digestible or available (relative) phosphorus, gestible or available phosphorus. The range phytase to the animal. equivalency values must be adjusted by the of tolerance ratio values is indicated in estimated digestibility and bioavailability parentheses. It is very important to enforce Unit of phytase activity added (relative) of the inorganic phosphorus a correct dietary ratio of calcium to phos- Commercially, there are two units used to sources that phytase replaces. In practical phorus, while ensuring dietary levels of cal- express phytase activity. One is FYT termed situations, a phosphorus equivalency value cium and phosphorus are adequate to meet fytase, and the other one is FTU termed fy- of 0.08% (0.8 g per kg diet) if it is based on the need of the animal. tase unit. These two units are derived from digestible phosphorus and of 0.10% (1.0 g assays performed under similar artificial per kg diet) if on available (relative) basis is Phytase, citric acid and conditions. These in vitro phytase activity recommended. Equivalency values may be metabolite assays are indispensable for controlling and derived from linear and curvilinear equa- Citric acid has been shown to improve phy- measuring the recovery of phytase in feed tions generated from responses of body tate phosphorus utilisation in broiler chick- manufacturing, processing and storage weight gain, bone mineralisation and di- ens, thus sparing a certain portion of dietary conditions. For simplicity, they both may be gested phosphorus obtained by feeding phosphorus. Boling et al. (2001) conducted referred to as phytase units (U). graded levels of phosphorus without phy- a broiler trial to evaluate the effect of citric Phytases are often dosed by a recom- tase addition and/or graded levels of added acid on growth and bone performance of 8- mended number of units of activity per kg phytase to a low-phosphorus diet. to 22-day-old broiler chickens fed corn-soy- into the diet. The bioefficacy of a phytase is bean meal diets, with two levels of available not indicated by its recommended dosage, Dietary Ca:P ratios phosphorus (0.20 or 0.45%) and four levels ie. One recommended at a higher activity Feeding calcium and phosphorus in prac- of citric acid (0, 2, 4 or 6%). A linear increase per kg of diet is not a reflection of poorer tice is illustrated in Figure 1. Under current in weight gain and tibia ash was observed quality than another dosed at lower activity practical conditions, dietary calcium levels when supplemental levels of citric acid were per kg diet, particularly referring to are always in excess of the requirement as increased in broiler chickens fed 0.20% Peniophora and Aspergillus phytases. Both limestone is preferred as a filler in least-cost available phosphorus. However, weight gain phytases catalyse the same reaction, name- feed formulation. and tibia ash in birds fed 0.45% available ly hydrolysis of phytic acid to release inor- When imbalances of dietary Ca:P ratios phosphorus were neither increased nor de-

18 www.AgriWorld.nl WORLD POULTRY - Elsevier Volume 18,No 8.'02 Figure 1: Feeding Ca & P in practise

creased by citric acid, regardless of what levels were fed. The mode of action of citric acid may be associated with its calcium complexing property and attributed to a reduction of the inhibitory effect of calci- um on phytate hydrolysis. Citric acid, a strong chelator of calcium, re- moves calcium from or decreases calcium binding to phytate, making it less stable (i.e., more soluble) and more susceptible to endogenous and exogenous phytase. Another possible mode of action of citric acid may be associated with its effects on intestinal pH. However, inconsis- tent findings are likely to dismiss this mode of action. Vitamin D metabolites such as 25-hydroxy-vitamin D3 (25-OH-D3) have been implicated in increasing phytate phosphorus utilisation. As such, it spares a certain portion of dietary phosphorus. Applegate and Angel (2002) hypothesise that the mode of action of 25-OH-D3 in im- proving phosphorus utilisation occurs indirectly through improving the rapid phase and slower phase of calcium uptake from the . At small intestinal pH, calcium can chelate to phytate, dra- matically reducing the phytate’s solubility. By removing a portion of calcium, phytate becomes more soluble and accessible to the hy- drolytic actions of endogenous or exogenous phytases. Secondarily, 25-OH-D3 may be assisting the hydrolytic action of phytase by reduc- ing the inhibitory effect of phosphorus ions from which the transloca- tion into the blood from intestinal mucosa is dependent upon vita- min D3. Applegate and Angel (2002) reported the sparing effect of phospho- rus when 12- to 21-day-old broiler chickens were fed diets containing supplemental phytase, citric acid and 25-OH-D3. In this experiment, three levels of phytase (0, 300 or 500 U/kg diet), three levels of citric µ acid (0, 1 or 2%) and three levels of 25-OH-D3 (0, 35 or 70 g/kg diet) were added to a low-phosphorus (0.2% nonphytate phosphorus), corn-soybean meal diets. The nonphytate phosphorus sparing effect for 500 U phytase/kg diet was 0.065%, whereas that for 35 and 70 µg 25-OH-D3 was 0.037 and 0.051% respectively. When added to the 500 µ U phytase/kg diet, 35 and 70 g 25-OH-D3 provided a sparing effect of 0.067 and 0.092%, respectively. The sparing effect when 500 U phy- µ tase/kg diet, 2% citric acid and 70 g 25-OH-D3 were used in combi- nation was 0.126%. Conclusion Factors affecting the response to phytase addition are largely ignored in commercial applications. By the same token, the potential of phy- tase is yet to be fully explored. Among the influencing factors, dietary calcium levels and calcium: phosphorus ratios are probably the two most important ones in terms of maximising the response to phytase. Managing these two factors aside from the other factors will allow greater solubility of phytate, rendering it to be more accessible to both endogenous and exogenous phytases. The response to phytase may be further enhanced by citric acid and 25-hydroxy-vitamin D3 where they both remove the inhibitory effect of calcium on phytate hydrolysis by phytase.□

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