Consequences of calcium interactions with phytate and phytase for poultry and pigs

Livestock Science 124 (2009) 126–141

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Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i

Consequences of calcium interactions with phytate and phytase for poultry and pigs Peter H. Selle a, Aaron J. Cowieson b, V. Ravindran c,⁎ a b c

Faculty of Veterinary Science, The University of Sydney, Camden, NSW 2750, Australia AB Vista, Marlborough, Wiltshire, SN8 4AN, United Kingdom Institute for Food, Nutrition and Human Health, Massey University, Palmerston North 4442, New Zealand

a r t i c l e

i n f o

Article history: Received 10 June 2008 Received in revised form 4 September 2008 Accepted 14 January 2009 Keywords: Calcium Phosphorus Phytate Phytase Pigs Poultry

a b s t r a c t Despite increasing practical experience and cascades of scientific reports on exogenous microbial phytases, several issues associated with their use remain unresolved because of the ambiguous and, at times, conflicting data that has been generated. One possible cause of these inconsistent outcomes is dietary calcium (Ca) levels, which are mainly derived from limestone. Thus the purpose of this review is to examine Ca interactions with dietary phytate and phytases, particularly exogenous, microbial phytases, and their consequences for poultry and pigs. The polyanionic phytate molecule has a tremendous capacity to chelate cations and form insoluble Ca–phytate complexes, which are refractory to phytase activity. Thus Ca–phytate complex formation along the gastrointestinal tract, where one phytate (IP6) molecule binds up to five Ca atoms, assumes importance and approximately one third of dietary Ca may be bound to phytate in digesta. Consequently, phytate limits the availability of both P and Ca as a result of insoluble Ca–phytate complex formation, the extent of which is driven by gut pH and molar ratios of the two components. It is accepted that Ca–phytate complexes are mainly formed in the small intestine where they have a substantial negative influence on the efficacy of mucosal phytase. However, exogenous phytases are mainly active in more proximal segments of the gut and lower pH levels, so their efficacy should not be influenced by Ca–phytate complexes in the small intestine. There is, however, data to indicate that Ca and phytate interactions occur under acidic conditions with the formation of soluble and insoluble Ca–phytate species, which could negatively impact on exogenous phytase efficacy. Also, Ca will tend to elevate gut pH because of limestone's very high acid binding capacity, which will favour Ca–phytate interactions and may influence the activity of exogenous phytases depending on their pH activity spectrum. The de novo formation of binary protein–phytate complexes that are refractory to pepsin hydrolysis may be fundamental to the negative impact of phytate on the digestibility of protein/amino acids. However, high dietary Ca levels may disrupt protein–phytate complex formation by interacting with both phytate and protein even at acidic pH levels, thereby influencing the outcomes of phytase amino acid digestibility assays. Finally, it is increasingly necessary to define the Ca and nonphytate-P requirements of pigs and poultry offered phytase-supplemented diets. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Phytate (myo-inositol hexaphosphate; IP6) is invariably present in pig and poultry diets at concentrations of approxi-

⁎ Corresponding author. E-mail address: [email protected] (V. Ravindran). 1871-1413/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2009.01.006

mately 10 g kg− 1. In order to dephosphorylate phytate and liberate the inherent phosphorus (P) component, inclusion of exogenous phytases in pig and poultry rations is an increasingly routine practice. The polyanionic phytate molecule also has a substantial capacity to chelate divalent cations, including calcium (Ca), to form mineral–phytate complexes. Moreover, insoluble Ca–phytate complexes are resistant to enzymatic hydrolysis by phytases (Taylor, 1965). Therefore,

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in theory, Ca is a limiting factor on phytate degradation and, alternatively, partial enzymatic hydrolysis of phytate by exogenous phytase should reduce the extent of Ca–phytate complex formation, thereby increasing the availability of Ca and phytate-bound P. For example, Pallauf et al. (1994) found that 700 FTU kg− 1 Aspergillus niger phytase increased Ca retention (0.73 versus 0.54) and P retention (0.71 versus 0.54) in young pigs offered diets based on wheat, barley and soyabean meal to similar extents. Also, Mroz et al. (1994) reported that 800 FTU kg− 1 A. niger increased Ca retention by 57.8% (0.464 versus 0.294) and P retention by 73.3% (0.551 versus 0.318) in pigs. Subsequently, Jongbloed et al. (2004) showed that 1000 FTU kg− 1 P. lycii phytase improved faecal digestibility of Ca by 47.7% (0.288 versus 0.195) and P digestibility by 78.0% (0.381 versus 0.214) in lactating sows. These outcomes are reflected in one supplier's recommendation that 500 FTU kg− 1 activity of A. niger phytase generates the equivalent of 1.00 g kg− 1 Ca and 1.15 g kg− 1 P in pig and broiler rations. However, the outcomes of phytase Ca equivalency studies that have been completed in broilers (Schöner et al., 1994; Mitchell and Edwards, 1996; Augspurger and Baker, 2004; Yan et al., 2006), turkeys and pigs (Kornegay et al., 1996) are inconsistent. Therefore, the release of Ca by microbial phytase merits attention as does the capacity of phytate to bind Ca and limit phytate degradation. Decades ago, Hill and Tyler (1954a) reported that Ca reduced wheat phytase efficacy, which was attributed Ca– phytate complex formation. Subsequently, Lei et al. (1994) reported that Ca, and/or ‘wide’ dietary Ca:P ratios, depressed responses to exogenous phytase in weaner pigs, which was also attributed to phytate complexing Ca. In broilers, Shirley and Edwards (2002) reported that Ca negatively impacted on phytase even at an inclusion rate of 6000 FTU kg− 1. Presently, it is accepted that increasing Ca concentrations, and Ca:P ratios, are deleterious to phytase efficacy in monogastric diets. Therefore, this review examines the probable consequences for pig and poultry nutrition of Ca interactions with dietary phytate and their impact on phytase efficacy. One objective is to consider the underlying mechanisms whereby Ca influences the efficacy of exogenous phytases because dietary Ca levels may be contributing to the inconsistent outcomes of phytase investigations that have been recorded in the literature. A second objective is to consider the underlying mechanisms and the extent to which phytate and phytase influence Ca availability so that appropriate adjustments may be made to formulations of phytasesupplemented experimental and practical diets. 2. Background In man and animals, Ca and P are essential nutrients for numerous biochemical pathways and skeletal integrity and the physiological roles of these two macro-minerals are inextricably linked (Wasserman, 1960). The aetiology of Osteomalacia, or rickets, is complex as it also involves vitamin D3 but phytate is a causative factor as it has the potential to limit both Ca and P availability (Bruce and Callow, 1934; Krieger et al., 1940; Mellanby, 1949; Bhaskaram and Reddy, 1979; Abugassa and Svensson, 1990). In this context, McCance and Widdowson (1942) provided a vivid example of Ca interacting with phytate and phytase. By activating the intrin-

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sic phytase activity of wheat bran, these workers reduced phytate in brown bread from 39.0 to 3.26 g kg− 1. The transition from standard to ‘dephytinised’ bread increased Ca absorption in human patients from an average of 36 to 165 mg day− 1, which was considered indicative of the rachitogenic properties of phytate. In addition, the negative impact of Ca on P absorption has implications for the treatment and prevention of Osteoporosis in humans (Heaney and Nordin, 2002). In animal nutrition, Ca and P requirements are largely met by dietary inclusions of limestone, inorganic P supplements such as dicalcium phosphate and, where permitted, meatand-bone meal. However, quite considerable amounts of P are present in plant-sourced feed ingredients but the majority of this P is phytate-bound and phytate-P is only partially, but variably, available to monogastrics. Phytate, the mixed salt of phytic acid (myo-inositol hexaphosphate; IP6) has been recognised for over a century (Hartig, 1855; Pfeffer, 1872). Phytate exists predominantly as IP6 in feed ingredients (Kasim and Edwards, 1998) with a P concentration of 282 g kg− 1 and a molecular weight of 660. The alternative, classic description of ‘phytin’ is synonymous with the calcium–magnesium salt of phytic acid (Plimmer, 1913). However, this may be misleading as, in the model proposed by Lott et al. (2000), phytate is typically present in feedstuffs as mineral–phytate complexes involving magnesium (Mg) and potassium (K) in which IP6 is bound to three Mg2+ and six K+ ions. Notionally, the phytate molecule carries a maximum of twelve negative charges and could potentially chelate six Ca atoms although the affinity of phytate is greater for certain other divalent cations, including zinc and copper (Maddaiah et al., 1964; Vohra et al., 1965). Nevertheless, Ca2+ is a dominant cation in diets and consequently de novo formation of insoluble Ca–phytate complexes in the gut at conducive pH levels assumes importance (Wise, 1983; Angel et al., 2002). This is illustrated in the equation proposed by Nelson (1984), where Ca requirements for broilers are calculated on the basis of dietary phytate-P concentrations Cað%Þ ¼ 0:6 þ ½phytate2Pð%Þ  1:1: This equation predicts an appropriate Ca level of 9.10 g kg− 1 in a 2.82 g kg− 1 phytate-P diet. It is likely that exogenous phytase degrades about 35% of dietary phytate at the ileal level in broiler chicks at standard inclusion rates (Selle and Ravindran, 2007). If so, this would effectively reduce phytate-P to 1.83 g kg− 1 and the Ca requirement to 8.02 g kg− 1. This in turn suggests phytase has a Ca equivalency value of 1.08 g kg− 1. Interestingly, an inherent premise in the above equation is that 5.1 atoms of Ca are bound by one phytate molecule. Phytase, the requisite enzyme to hydrolyse phytate, occurs widely throughout nature and phytase activity was first detected a century ago (Suzucki et al., 1907). Phytase is intrinsically present in certain plant-sourced feed ingredients (Godoy et al., 2005) and wheat, in particular, possesses ‘plant’ phytase activity (Anderson, 1915). However, plant phytase is heat-labile and may be eliminated when diets are steampelleted at high temperatures in excess of 85 °C (Jongbloed and Kemme, 1990). Gut microflora generates phytase activity, predominantly in the large intestine (Sandberg et al., 1993;

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Kerr et al., 2000) and while this may have limited nutritional and ecological importance, hindgut fermentation, particularly in pigs, confounds total tract assessments of phytate degradation. The presence of endogenous phytase activity was first demonstrated in rats by Patwardhan (1937) and, more recently, it has been shown that the small intestinal mucosa generates endogenous phytase activity in pigs (Hu et al., 1996) and poultry (Maenz and Classen, 1998). Nevertheless, the contribution to phytate hydrolysis in pigs and poultry by mucosal phytase and phosphatase is usually disregarded, perhaps because standard dietary Ca levels have a substantial negative influence on phytate degradation by endogenous enzymes. Exogenous, microbial phytases are mainly active in the stomach of pigs and fore-stomach of poultry where acidic pH increases substrate solubility and phytate is more susceptible to degradation (Campbell and Bedford, 1992). The dietary inclusion of fungal and bacterial phytases has been an option for nutritionists since 1991 and their acceptance has accelerated recently (Bedford, 2003) so that probably over half the pig and poultry diets globally contain an exogenous phytase. By enhancing the digestibility of phytate-P and permitting lower dietary P levels, phytase reduces the excretion of undigested and excess P in pigs and poultry by in the order of 30% (Simons et al., 1990). This is environmentally beneficial as phosphorus pollution is a hazard to aquatic ecosystems because P is the prime cause of eutrophication in fresh-water reserves (Sharpley, 1999; Sharpley et al., 1995; Sims et al., 1998; Correll, 1998, 1999). Increasing legislation designed to curb P pollution has contributed to the growing acceptance of phytase feed enzymes. A secondary issue is that global P reserves are not renewable and their depletion would be delayed by the inclusion of phytase in monogastric diets (Abelson, 1999; Mullaney et al., 2000). If, as claimed, 500 FTU kg− 1 phytase generates 1.15 g kg− 1 P in pigs and broilers then phytase corresponds to 6.4 g kg− 1 dicalcium phosphate in terms of dietary P. Inorganic P sources are escalating in price and, simultaneously, the cost of inclusion of microbial phytases is being eroded, thus phytase is becoming an increasingly attractive P source. This has been a key factor in the acceptance of phytase, as has the dietary exclusion of meat-and-bone meal in relevant countries. Additionally, although this issue remains controversial, phytase may enhance protein and energy utilisation in pigs (Selle and Ravindran, 2008) and poultry (Selle and Ravindran, 2007). Consequently, some nutritionists elect to assign amino acid and energy matrix values to exogenous phytases in leastcost ration formulations for pigs (Radcliffe et al., 2006) and poultry (Shelton et al., 2004), which further enhances their economic value. Nutritionists have displayed interest in the potential use of phytase for decades, particularly in poultry (Nelson, 1967; Rojas and Scott, 1969). However, in addition to phytate-P availability, this interest was also prompted by the capacity of phytate to reduce Ca availability (Nelson, 1984; Nelson and Kirby, 1987). Following the landmark Simons et al. (1990) study, a flood of investigations into various facets of phytase supplementation of pig and poultry diets have been reported. Essentially, these studies support the capacity of phytase to enhance phytate-P availability but, beyond this arena, the outcomes often lack consistency and clarity.

Undoubtedly, the dietary substrate level is one factor that has contributed to this situation. For example, in two Australian surveys (Kim et al., 2002; Selle et al., 2003), phytate in wheat ranged from 3.7 to 11.4 g kg− 1 and obviously such variations would make substantial differences to phytate concentrations in complete wheat-based diets. However, mineral–phytate complexes and Ca–phytates in particular, reduce phytase efficacy (Nelson, 1984; Angel et al., 2002) so it follows that dietary Ca level is another factor that has contributed to ambiguous outcomes from experiments involving exogenous phytases. In the above two wheat surveys, phytate-P levels were determined by a ‘ferric chloride-precipitation method’, based on the Heubner and Stadler (1914) principle, from which phytate concentrations were derived. While more refined analytical methods that can differentiate phytate esters have since been developed, as discussed by Selle and Ravindran (2007), phytate determinations are not straightforward. As a consequence, stated levels of phytate or phytate-P should be treated cautiously and this unsatisfactory situation is reflected in apparently anomalous results of phytate concentrations in feedstuffs, diets, digesta and excreta that have been published in the literature on occasion. Also, phytase (fytase) activity is classically defined as FTU kg− 1 where one FTU is the enzyme activity that liberates 1 μmol inorganic P from 0.0051 mol L− 1 sodium phytate at pH 5.5 and 37 °C per minute (Engelen et al., 1994). Alternative, but essentially similar, definitions are used; however, for the sake of consistency phytase activity is expressed as FTU kg− 1 throughout this review. 3. Dietary calcium levels and calcium:phosphorus ratios It is accepted that exogenous phytases in pig and poultry diets are advantaged by relatively low dietary Ca levels and ‘narrow’ Ca:P ratios and the probable genesis of this is the Lei et al. (1994) study. Weaner pigs were offered P-inadequate (3.11 g kg− 1 total P), maize–soy diets with Ca:P ratios of 1.62 to 3.07 that contained 4 or 8 g kg− 1 Ca and 750 or 1200 FTU kg− 1 A. niger phytase. Over a 30-day feeding period, higher Ca levels substantially depressed weight gain (32.0%), feed intake (16.2%) and feed efficiency (18.8%) in pigs receiving phytase-supplemented diets. The interpretation of Lei et al. (1994) was that higher Ca levels and wider Ca:P ratios depressed exogenous phytase efficacy, which was attributed to Ca progressively precipitating phytate in ‘extremely insoluble’ Ca–phytate complexes in the intestine. However, a superior trial design would have included non-phytase supplemented diets to determine the impact of Ca per se in this context. Several, generally supportive studies in pigs (Lantzsch et al., 1995; Qian et al., 1996b; Liu et al., 1998; Li et al., 1999; Brady et al., 2002), broilers (Sebastian et al., 1996; Qian et al., 1997) and turkeys (Qian et al., 1996c) have been published. Also, Liu et al. (2000) reported that increasing Ca:P ratios from 1.0 to 1.5:1 reduced ileal absorption of P from 4.5 to 2.2 g day −1 in pigs offered low-P, phytase-supplemented diets. In 42day old broilers, Aksakal and Bilal (2002) found that phytase increased P retention by 8.5% in diets with a Ca:P ratio of 2:1, but P retention was increased to 39.8% with Ca:P ratios of 1:1. Zyla et al. (2000) reported that phytase increased 21-day

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weight gain by 8.4% in broilers offered diets with 1.93 Ca:P ratios, as opposed to increases of 13.4% and 19.6% in diets with narrower Ca P ratios 1.44 and 1.68. Alternatively, Van der Klis et al. (1997) found that increasing dietary Ca levels from 30 to 40 g kg− 1 in nonsupplemented diets reduced ileal phytate degradation from approximately 33% to 9% in laying hens. However, in diets supplemented with 500 FTU kg− 1 phytase, the reduction in phytate degradation induced by Ca was relatively modest, from approximately 76% to 64%. In broilers, Singh and Sikka (2006a,b) investigated the effect of four Ca:P ratios ranging from 1:1 to 2:1 but significant responses to phytase were observed at all Ca:P ratios for weight gain, feed intake and retention of Ca, P and nitrogen. Recently, Adeola et al. (2006) investigated this aspect in weaner pigs offered maize–soy diets containing 4.0 g kg− 1 total P and 0.8 g kg− 1 non-phytate P. In a 3 × 2 factorial array of dietary treatments, limestone was added to provide 4.9, 6.0 and 7.1 g kg− 1 Ca, or Ca:P ratios 1.8, 1.5 and 1.2, without and with 1000 FTU kg− 1 Escherichia coli-derived phytase. As a main effect, decreasing Ca:P ratios from 1.8 to 1.2 significantly enhanced weight gain by 17.3% and feed efficiency by 35.2%. However, irrespective of the Ca:P ratio, phytase significantly increased weight gain by 30.0% and feed efficiency by 21.3%. There were no significant treatment interactions between phytase and Ca:P ratio for weight gain and feed efficiency. Moreover, in young broilers, Driver et al. (2005a) reported that 1200 FTU kg− 1 A. niger phytase was more effective in maize–soy diets containing 8.6 g kg− 1 Ca, 4.5 g kg− 1 total P and 2.0 g kg− 1 nonphytate P (Ca:P ratio of 1.91) than in 4.7 g kg− 1 Ca, 5.0 g kg− 1 total P and 2.4 g kg− 1 nonphytate P (Ca:P ratio 0.94) diets. Phytase induced increases in weight gain (64.5 versus 12.5%), feed intake (54.4 versus 10.4%) and tibia ash (41.6 versus 9.9%) were noticeably more pronounced in the higher Ca diets and the treatment interactions were highly significant. Also, the treatment interaction in feed efficiency (6.2 versus 1.6%) approached significance. Predictably, these authors concluded that much of the published data concerning the efficacy of phytase at different Ca:P ratios is misleading, that phytase efficacy is a complex function of dietary Ca, total P and phytate-P concentrations, and that Ca reactions with inorganic P, which may lead to the flocculent precipitation of calcium orthophosphate [Ca3(PO4)2] merit more attention. While the Lei et al. (1994) study, and similar studies, is open to criticism, relevant questions as to the influence of Ca on phytate degradation by phytase in pigs and poultry have been raised. 4. Interactions between phytate and calcium 4.1. Formation of calcium–phytate complexes The extent to which Ca exists as mineral–phytate complexes in monogastric diets is limited by the relatively low Ca concentrations in relevant feed ingredients (Table 1) and the inherent structure of phytate, as defined by Lott et al. (2000). Therefore, the de novo formation of Ca–phytate complexes along the gastrointestinal tract of pigs and poultry is more important. Ca–phytate complex formation is influenced by molar ratios of the constituents and gut pH and their reduced solubility means they are less readily degraded by phytases

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Table 1 Typical concentrations of calcium, total phosphorus, phytate–phosphorus, proportion of phytate-P to total P, and phytate in key feed ingredients. Feed ingredient

Total P Phytate-P Phytate-P/ Phytate Ca (g kg− 1) a (g kg−1) b (g kg− 1) b total P (%) b (g kg− 1) c

Barley Maize Sorghum Wheat Canola meal Cottonseed meal Soyabean meal Rice bran Wheat bran

0.30 0.20 0.40 0.50 6.80 1.50 2.70 0.50 1.40

a b c

3.21 2.62 3.01 3.07 9.72 10.02 6.49 17.82 10.96

1.96 1.88 2.18 2.19 6.45 7.72 3.88 14.17 8.36

61 72 73 72 66 77 60 80 76

7.0 6.7 7.7 7.8 22.9 27.4 13.8 50.3 29.6

NRC (1994). Selle and Ravindran (2007). Calculated on the basis that phytate contains 282 g kg− 1 P.

residing in the gastrointestinal tract (Wise, 1983). However, specific investigations into Ca–phytate interactions at molar ratios found in typical diets and across physiologically relevant pH ranges are limited. Importantly, pH is crucial to the solubility of Ca–phytate complexes and pH in digesta ranges from highly acidic in the stomach and proventriculus to approaching neutrality in the small intestine. Under in vitro conditions, Wise and Gilburt (1981) found that Ca–phytate was soluble below pH 4, but precipitation was observed at pH 5, and this finding was emphasised in the pivotal review by Wise (1983). Nash et al. (1998) reported that solid calcium phytate contained an average of 4.62 Ca atoms per molecule at pH 7 and Evans et al. (1983) proposed that an insoluble Ca–phytate complex comprises Ca5K2–phytate. Martin and Evans (1986) completed a detailed investigation into the effect of pH on Ca–phytate binding but did not detect any interactions below pH 5. Several studies (Evans and Pierce, 1981; Grynspan and Cheryan, 1983; Oberleas and Chan, 1997) generally support this conclusion that pH 5 is pivotal to Ca–phytate complex formation. Marini et al. (1985) found an average molar ratio of 4.93:1 in Ca–phytate complexes. Pig and poultry diets typically contain 10 g kg− 1 of both Ca and phytate; then if one phytate molecule binds five Ca atoms in the gastrointestinal tract approximately one third of dietary Ca would be present as Ca–phytate complexes. Phytate is predominantly present as IP6 in feedstuffs (Lott et al., 2000). However, Kaufman and Kleinberg (1971) reported that pH 5.4 was critical to the precipitation of Ca complexes across a range of phytate esters (IP1 to IP5), where lesser esters remained soluble at higher pH values. Lower phytate esters have a disproportionately diminished capacity to bind Ca (Luttrell, 1993), which was demonstrated by Lonnerdal et al. (1989) in young rats. IP6 reduced Ca absorption by 17% but the effect of IP5, IP4 and IP3 on Ca uptake was negligible. The data considered suggests that Ca–phytate complexes are not likely to precipitate at less than pH 5.0. In contrast, however, Graf (1983) concluded that phytate exhibits a high affinity for Ca2+ over a wide pH range following Ca2+-selective potentiometry investigations. While this affinity increased with pH, Ca–phytate chelates were formed at acidic pH as low as 2.0 and two soluble Ca2+–phytate species were detected.

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Subsequently, Marini et al. (1985) reported Ca–phytate binding from pH 2.0 to 12.0 and Champagne (1987) also found soluble Ca2+–phytates at pH 2.4 to 5.9. Furthermore, Graf (1983) suggested that chelation of Ca2+ at low pH indicates that substantial amounts of Ca will be bound by phytate in the stomach. This contrasts to the conventional view that mineral– phytate complex formation occurs at the relatively high pH of the small intestine and phytate will preferentially bind proteins at more acidic pH levels (Wise, 1983). 4.2. Calcium–phytate complexes and phytase activity Ballam et al. (1985) stressed the importance of Ca and Ca– phytate complexes on phytate degradation and the liberation of phytate-P. In support of this, Hill and Tyler (1954a) found that Ca reduced phytate hydrolysis by wheat phytase by 22% in an in vitro system. Also, Gillis et al. (1957) reported that radioactively-labeled Ca–phytate bioavailability was negligible in comparison to inorganic P (Na2HP32O4) as assessed by P retention in tibia ash of broiler chicks and turkey poults. More recently, Plumstead et al. (2007) demonstrated that increasing dietary Ca from 4.7 to 11.6 g kg− 1 in broiler diets linearly decreased ileal phytate-P digestibility by 71%, from 0.201 to 0.059. Instructively, Pang and Applegate (2007) found Ca solubility was only 11% in the duodenum and jejunum of chickens (pH 6.26) offered diets containing 9.0 g kg− 1 Ca and 9.22 g/kg phytate, presumably as a consequence of Ca–phytate aggregations. The site of insoluble Ca–phytate complex formation is relevant to the efficacy of exogenous phytases where the conventional view is that complex formation occurs in the small intestine (Wise, 1983). Crucially, therefore, the major site of A. niger phytase activity is in the stomach of pigs (Jongbloed et al., 1992; Yi and Kornegay, 1996; Pagano et al., 2007) and the crop in poultry (Liebert et al., 1993; Takemasa et al., 1996; Kerr et al., 2000). Consequently, if Ca–phytate complexes are not formed until digesta reaches the small intestine, then Ca would not impede exogenous phytase activity more proximally in the stomach or fore-stomach. Thus, the real possibility that Ca and phytate interactions occur at acidic pH, subject to Ca:phytate molar ratios, has distinct implications in pigs and poultry in relation to exogenous phytase efficacy. Importantly, Mroz et al. (1992) found that the infusion of 1260 FTU kg− 1 A. niger phytase into the duodenum of pigs offered maize–soy diets did not tangibly alter ileal phytate degradation. The modest increase in phytate degradation along the small intestine could indicate (i) the presence of refractory Ca–phytate complexes, (ii) an excessive intestinal pH relative to the activity spectrum of phytase and/or (iii) its inactivation by proteolytic enzymes. However, it is possible that bacterial phytases are more resistant to denaturation by endogenous proteolytic enzymes than fungal phytases (Igbasan et al., 2000; Onyango et al., 2005). In poultry the position is different, Shafey et al. (1991) reported a pH of 4.89 in crop digesta in broilers offered practical diets so there is a greater likelihood of Ca–phytate interactions in the proximal gastrointestinal tract in poultry. Increased lighting intervals probably reduce digesta retention in the crop (Hoopaw and Goodman, 1976) and provide less scope for phytate degradation by phytase. Interestingly,

Bedford et al. (2007a) found that phytase-induced increases in growth rates diminished as lighting intervals lengthened, from an analysis of 77 broiler studies. Thus, it would appear that phytase is advantaged by reduced lighting regimes and increased crop retention despite the likelihood that this would also favour Ca–phytate interactions. Limestone, the dominant source of Ca in pig and poultry diets, has an extremely high acid binding capacity of 15,044 meq kg− 1 at pH 3 (Lawlor et al., 2005). Thus Ca, as limestone, will tend to increase digesta pH along the gut and Shafey et al. (1991) reported that increasing dietary Ca from 10.7 to 25.3 g kg− 1 increased crop pH from 4.89 to 5.32. It follows that Ca–phytate interactions would be favoured by higher pH coupled with a greater Ca:phytate molar ratio. Conversely, where glutamic acid reduced crop pH from 6.0 to 5.4 microbial phytase efficacy was enhanced in respect of bone mineralisation (Murai et al., 2001), possibly via decreased Ca–phytate complex formation and/or increased Mg–K–phytate solubility. Additionally, gut pH will directly influence exogenous phytase activity depending on the pH activity spectrum of a given enzyme. For example, A. niger phytase has a bi-phasic pH activity spectrum with one peak at pH 3.0 and the more pronounced peak at pH 5.5; moreover, an increase to pH 6.0 results in a substantial decline in phytase activity (Engelen et al., 1994). Thus the impact of Ca as limestone on gut pH is relevant. By increasing dietary Ca levels from 9.0 to 13.0 g/kg with limestone, McDonald and Solvyns (1964) increased digesta pH from 5.6 to 6.1 along the length of the small intestine and increased Ca levels were negatively correlated to broiler growth rates (Table 2). Shafey (1999) subsequently recorded a similar limestone-induced pH increase (5.68 to 6.24) in small intestinal digesta and, as shown in Table 5, Shafey et al. (1991) found that increased dietary Ca (25.3 versus 10.7 g kg− 1) significantly increased crop and ileum pH. Therefore, additional Ca as limestone has the potential to increase insoluble Ca– phytate complex formation by increasing both gut pH and Ca: phytate molar ratios. In respect of mucosal phytase efficacy in broilers, Tamim et al. (2004) reported a phytate ileal degradation coefficient of 0.692 in non-supplemented, maize–soy broiler diets containing 2.8 g kg− 1 phytate-P but only 2.0 g kg− 1 Ca. However, an increase in dietary Ca to 7.0 g kg− 1 resulted in a substantial 63% decrease in phytate degradation (0.254 versus 0.692). A similar 72% reduction in phytate degradation by mucosal phytase was reported earlier following the addition of 5.0 g kg− 1 Ca (Tamim and Angel, 2003). Presumably, more extensive Ca–phytate complex formation was major causative factor in these pronounced reductions in

Table 2 Influence of dietary Ca levels on apparent ileal digestibility of phytate-P, total P and nonphytate P in broilers (adapted from Tamim et al., 2004). Dietary Ca (g kg− 1)

2.0 7.0 Difference (%)

Phytate-P (2.8 g kg− 1)

Total P (4.1 g kg− 1)

Nonphytate P (1.3 g kg− 1)

AID

g kg− 1

AID

g kg− 1

AID

g kg− 1

0.692 0.254 0.438 (63.3)

1.94 0.71 1.23 (63.4)

0.679 0.294 0.385 (56.7)

2.78 1.21 1.57 (56.5)

0.646 0.385 0.261 (40.4)

0.84 0.50 0.34 (40.5)

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phytate degradation. However, even if the additional Ca bound phytate to the maximum extent this would not, theoretically, fully account for the observed reductions in phytate degradation in these two experiments, which raises the possibility that other mechanisms may be operative (Table 3). Ca has the capacity to interact with inorganic P in the gut lumen in addition to phytate-P (Hurwitz and Bar, 1971). Tamim et al. (2004) reported that 5 g kg− 1 Ca reduced the ileal digestibility of phytate-P by 63% but additional Ca also reduced the digestibility of total P by 57%. Thus it may be deduced that Ca reduced inorganic or nonphytate P digestibility by approximately 40% (Table 4). Thus it appears that Ca was binding with both phytate and inorganic P to form either Ca–phytate complexes or calcium phosphates and it is relevant that calcium orthophosphate is only sparingly soluble in excess of pH 5.0 (Holt et al., 1925). This position may be represented as follows: Ca–phytate complexes ⇐ phytate þ Ca þ inorganicP ⇒ calcium phosphates; Ca5  K2  IP6 ⇐ IP6 þ Ca þ PO4 ⇒ Ca3 ðPO4 Þ2 : The direction of the proposed equations may be driven by relative concentrations of phytate and inorganic P; however, it is likely that Ca has a greater affinity for phytate than orthophosphate (Gosselin and Coghlan, 1953) Also, increasing Ca:P ratios from 1:1 to 2:1 has been shown to depress phytate-P availability to a greater extent than P from dicalcium phosphate in broilers (Harms et al., 1962). Tamim and Angel (2003) investigated the impact of Ca on phytate hydrolysis by microbial phytase in an in vitro system. Additional Ca reduced the liberation of P from sodium phytate by A. niger phytase from ∼ 350 to ∼175 μg P unit− 1 phytase at pH 6.5. However, at pH 2.5, while the magnitude of phytate hydrolysis was substantially greater, Ca induced a similar relative reduction in degradation from ∼ 1250 to ∼625 μg P unit− 1 phytase, which suggests Ca was interacting with sodium phytate at pH 2.5. In broilers, Tamim et al. (2004) determined the effect of Ca on the efficacy of two fungal phytases (A. niger or Peniophora lycii), as shown in Table 5. At the lower Ca level there was no difference between the two

Table 3 Influence of phytase supplementation and dietary Ca levels on apparent ileal digestibility (AID) of calcium and phytate-P in broilers (adapted from Tamim et al., 2004). Treatment

Ca

Phytate-P

Phytase1 (FTU kg− 1)

Calcium (g kg− 1)

AID coefficient

Digestible Ca (g kg− 1)

AID coefficient

0 500 500 0 500 500

2.0 2.0 2.0 7.0 7.0 7.0

0.336d 0.489b 0.415c 0.463bc 0.589a 0.581a

0.559c 0.811c 0.689c 2.991b 4.024a 3.803a

0.692b 0.795a 0.762ab 0.254e 0.589c 0.449d

A B A B

a–e Means within a column with no common superscript are significantly different (P b 0.05). 1 A. Aspergillus niger phytase B. Peniophora lycii phytase. At 7.0 g kg− 1 Ca, A. niger phytase generated 1.033 g kg− 1 ileal digestible Ca. At 7.0 g kg− 1 Ca, P. lycii phytase generated 0.812 g kg− 1 ileal digestible Ca.

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Table 4 Influence of dietary Ca levels on weight gain and pH of small intestinal digesta in chickens (adapted from McDonald and Solvyns, 1964). Ca level (g kg− 1)

Added limestone (g kg− 1)

Weight gain a (g)

Small intestinal pH

9.0 11.0 13.0 17.0 25.0 LSD b (P b 0.05)

0 5.0 10.0 20.0 40.0

77.0 77.5 76.0 70.8 63.5 4.0

5.6 5.8 6.1 6.1 6.0 0.5

a From the tabulated means there is a significant negative correlation between dietary Ca level and weight gain (r = − 0.984; P b 0.01). b Least significant difference.

phytases; however, A. niger phytase degraded 33.5% of dietary phytate at the higher Ca level as opposed to 19.5% phytate degradation by P. lycii phytase. This indicates Ca has a greater negative impact on P. lycii phytase efficacy and the researchers suggested that this may be related to the differentiating influence of limestone and gut pH. Phytate is mainly present in feed ingredients as Mg– phytate complexes with a molar ratio in the order of 4.4:1 at pH 6 (Evans and Pierce, 1982). Clearly the effect of gut pH on their solubility is relevant and Cheryan et al. (1983) reported that Mg–phytate complexes were soluble below pH 5 but rapidly became insoluble as pH levels increased above pH 5. However, with increasing Mg:phytate molar ratios reduced solubility was observed at lower pH values. It follows that high limestone levels may reduce the solubility of Mg–phytate complexes and their susceptibility to enzymic degradation in the crop of broilers. It is noteworthy that the effect of limestone particle size on phytase efficacy has been evaluated. Depending on limestone particle size, Manangi and Coon (2007) reported that phytase released from 567 to 656 μg P FTU− 1 at pH 2.5 and 367 to 458 μg P FTU− 1 at pH 6.5. However, the minimum P release was associated with the smallest particle size (28 μm) at both pH levels. Based on both in vitro and broiler studies, it was concluded that limestone, with a small particle size and high solubility, limited phytate hydrolysis by facilitating Ca–phytate complex formation. While peripheral to this review, the effect of maize particle size in this context has been investigated by Kasim and Edwards (2000) and Kilburn and Edwards (2001).

Table 5 Effect of Ca on pH of digesta along the gastrointestinal tract of broiler chickens (adapted from Shafey et al., 1991). Site

pH 10.7 g kg− 1 Ca

25.3 g kg− 1 Ca

Crop a Proventriculus Gizzard Duodenum Jejunum Ileum b Caecum

4.89 1.98 3.14 5.53 6.06 6.62 6.48

5.32 2.11 3.09 5.76 5.97 7.39 6.73

a b

P b 0.05. P b 0.01.

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Of relevance is that β-propeller phytase is active at neutral and alkaline pH (Greiner et al., 2007). Optimal activity is evident at pH 6–8 and β-propeller phytase targets calcium– phytate complexes (Fu et al., 2008) and may have the capacity to hydrolyse Ca-bound phytate completely (Shin et al., 2001). It would appear, therefore, that the inclusion of this particular phytase in pig and poultry diets may hold promise. 4.3. Direct impact of calcium on phytase activity There is the suggestion that Ca may inhibit phytase activity in vitro (Qian et al., 1996a). It was considered that this partial, competitive inhibition of A. niger phytase stemmed from Ca directly repressing phytase activity by competing for active sites of the enzyme. Scheuermann et al. (1988) reported that Ca inhibited the hydrolysis of Ca-phytate by wheat phytase by up to 65%. Applegate et al. (2003) reported that increasing Ca from 4.0 to 9.0 g kg− 1 in maize–soy diets depressed ileal phytate degradation by 44.4% (0.220 versus 0.396) in broilers. However, while this was associated with reduced mucosal phytase kinetics in brush-border vesicles, this reduction was of a lower order than the reduction in phytate degradation. Without dismissing the possibility, there is little tangible evidence that Ca directly inhibits exogenous phytase activity. 5. Phytase and calcium availability 5.1. Calcium equivalency of exogenous phytase Numerous studies have been completed in pigs (Selle and Ravindran, 2008) and poultry (Selle and Ravindran, 2007) to determine the P equivalency of exogenous phytase. However, relatively few parallel studies with Ca have been completed although it is accepted that phytase liberates phytate-bound Ca and perhaps one third of dietary Ca is present as Ca–phytate complexes in small intestinal digesta. However, Augspurger and Baker (2004) recently reported that 500 FTU kg− 1 E. coli-derived phytase in Ca-deficient (4.8 g kg− 1) maize–soy broiler diets released an average of 0.90 g kg− 1 Ca on the basis of tibia ash. Earlier, Kornegay et al. (1996) completed Ca equivalency studies in pigs and turkeys. In the first pig experiment, either phytase (200, 400, 600 FTU kg− 1) or Ca as limestone (4.6, 5.5, 6.4 g kg− 1) were added to a basal diet containing 3.7 g kg− 1 Ca. Based on weight gain, total tract Ca digestibility and percentage rib ash, 500 FTU/kg was equivalent to 1.08 g kg− 1 Ca. While the second pig study was very similar, the equivalency value of 0.38 g kg− 1 Ca was markedly lower. In turkeys, five levels of phytase (125, 250, 375, 500, 625 FTU kg− 1) or five levels of Ca as limestone (6.0, 6.7, 7.4, 8.1, 8.8 g kg− 1) were added to a 5.3 g kg− 1 Ca basal diet. It was estimated that 500 FTU kg− 1 phytase was equivalent to 0.87 g kg Ca− 1, based on weight gain, feed efficiency and total tract Ca digestibility. In broilers, Schöner et al. (1994) added graded inclusion levels of either Ca as limestone (0.75, 1.50, 2.25 g kg− 1) or A. niger phytase (250, 500, 1000 FTU kg− 1) to a basal diet, which contained 4.0 g/kg Ca, 6.0 g kg− 1 total P and 2.3 g kg− 1 phytateP. From the tabulated means (Table 6), it may be calculated that 500 FTU kg− 1 phytase is equivalent to 0.444 g kg− 1 Ca, based on the mean of responses in live-weight gain and toe ash. This low equivalency value implies that relatively small amounts

Table 6 Weight gain and toe ash data from a phytase Ca equivalency study in broilers offered basal diets containing 4.0 g kg− 1 Ca and 6.0 g kg− 1 P (adapted from Schöner et al., 1994). Addition rates Ca (g kg− 1)

Phytase (FTU kg− 1)

0 0 0 0 0.75 1.50 2.25

0 250 500 1000 0 0 0

Weight gain (g bird− 1)

Toe ash (mg)

519d 540cd 552cd 557bcd 579bc 598ab 640a

22.1d 23.0cd 23.8bcd 24.2bc 24.3bc 24.9b 27.8a

a–d Within a column, mean values not sharing a common superscript are significantly different (P b 0.05).

of Ca were bound by phytate in the experimental diets. However, Farkvam et al. (1989) found that increasing dietary Ca concentrations in broiler diets increased the amount of Ca bound by phytate. Thus the intentionally low basal Ca levels in the Schöner et al. (1994) study may reduce the amount of Ca bound by phytate and explain the low Ca equivalency value, which is less than half that usually incorporated into nutrient matrixes for phytase feed enzymes. In contrast to the above studies, Yan et al. (2006) concluded that phytase releases minimal amounts of Ca in broilers. These workers added 1000 FTU kg− 1 phytase to maize–soy diets containing three levels of Ca and eight levels of nonphytate P. It is possible that widely varying Ca:P ratios (and Ca:phytate ratios) in the complicated trial design contributed to the outcome. Mitchell and Edwards (1996) also concluded that exogenous phytase did not reduce Ca requirements in young broilers in a study where Ca:P ratios ranged widely from 0.86 to 2.30 and the diets contained a modest level of 2.02 g kg− 1 phytate-P. Perhaps both factors are relevant to the lack of response to phytase in terms of Ca. 5.2. Impact of phytase on ileal calcium digestibility Arguably, phytase Ca equivalency studies are confounded by mandatory, low dietary Ca levels and the impact of phytase on ileal Ca digestibility may be more meaningful. Not all the relevant reports in the literature are reviewed herein. However, in pigs, O'Quinn et al. (1997) reported that 500 FTU/kg phytase increased ileal digestibility of Ca by 6.4% (0.633 versus 0.595) in grower pigs offered sorghum-based diets. The diets contained 4.0 g kg− 1 so the study implies that phytase generated 0.152 g kg− 1 ileal digestible Ca, which is not a pronounced response. Traylor et al. (2001) assessed the effect of A. niger phytase on the ileal digestibility of Ca in soyabean mealbased diets containing 6.0 g kg− 1 Ca in grower pigs. Phytase (1500 FTU kg− 1) increased AID of Ca by 9.0% (0.692 versus 0.635) and TID by 8.2% (0.750 versus 0.693), which is an approximate 0.516 g kg− 1 release of digestible Ca. Guggenbuhl et al. (2007) compared the impact of three microbial phytases on total tract digestibility of P and Ca in growing pigs. At standard inclusions, the three phytases enhanced P and Ca digestibility to similar extents. Overall, phytases increased P digestibility by 84.9% (0.457 versus 0.247) and Ca digestibility by 15.3% (0.664 versus 0.576),

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which would suggest that phytase generated 0.800 g kg− 1 P and 0.638 g kg− 1 Ca on a total tract basis. Igbasan et al. (2001) assessed the capacity of fungal and bacterial phytases to enhance Ca bioavailability in pigs and poultry. In broilers, 500 FTU kg− 1 phytase numerically increased ileal Ca digestibility by 24.4% (0.536 versus 0.431). Surprisingly, this difference was not significant but it suggests that phytase liberated 1.05 g kg− 1 Ca from 10.0 g kg− 1 diets. In young pigs offered diets containing 8.0 g kg− 1 Ca, both phytases significantly improved Ca retention in a balance study by an average of 19.2% (0.665 versus 0.558), which suggest a 0.856 g kg− 1 Ca release following phytase inclusion. In poultry, Ravindran et al. (2006) determined the effects of phytase on apparent ileal Ca digestibility in maize–soy broiler diets containing 8.2 g kg− 1 Ca. Graded additions (0, 500, 750, 1000 FTU kg− 1) of an E. coli-derived phytase increased ileal digestibility coefficients of Ca (0.389 versus 0.352) by 10.5% overall, or phytase generated an average of 0.303 g kg− 1 ileal digestible Ca. More recently, Ravindran et al. (2008) reported that 500 FTU kg− 1 E. coli-derived phytase increased ileal Ca digestibility by 27.0% (0.329 versus 0.259) in maize–soy diets containing 7.8 g kg− 1 Ca, thus generating 0.546 g kg− 1 ileal digestible Ca. These and other studies indicate that phytase has the capacity to enhance Ca digestibility in pigs and poultry. However, because the digestibility of Ca is inherently poor, the amount of ileal digestible Ca generated by phytase is considerably greater when expressed as dietary Ca levels. 5.3. Mechanisms whereby phytase enhances calcium availability Essentially, exogenous phytases enhance Ca absorption in the small intestine by the partial hydrolysis of phytate to lower phytate esters in more proximal segments of the gut. Lower phytate esters have a disproportionately reduced capacity to chelate Ca, so insoluble Ca–phytate complex formation is diminished and Ca availability is correspondingly enhanced. However, it is also possible that phytase facilitates the intestinal uptake of Ca. Calcium absorption across the intestinal epithelium is stimulated by 1,25-dihydroxyvitamin D3 but it is also influenced by Na, which may alter Ca transport at the brush border by altering electrical gradients and at the basolateral membrane via intracellular Ca exchange (Favus, 1985). Recently, it has been demonstrated that phytate has the capacity to drag sodium (Na) into the gut and suggested that this may impede small intestinal uptakes of amino acids and other nutrients by compromising Na-dependent transport systems (Cowieson et al., 2004; Selle et al., 2007; Ravindran et al., 2008). Instructively, Kennefick and Cahman (2000) found that sodium phytate significantly reduces transepithelial Ca transport and Ca uptake by Caco-2 cells. This is consistent with the possibility that phytate is impeding intestinal uptakes of Ca by compromising Na-dependent transport systems and, by ameliorating Na secretion into the gut, phytase may enhance Ca absorption. 6. Calcium and the ‘protein effect’ of phytate and phytase One of the most pertinent unresolved issues is the capacity of exogenous phytase to increase the ileal digestibility of

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amino acids, and protein utilisation, in pigs and poultry. Some researchers maintain that phytase does not enhance protein utilisation (Adeola and Sands, 2003). However, in broiler studies where acid insoluble ash or titanium oxide has been used as inert dietary markers, phytase has consistently increased ileal digestibility of amino acids. In contrast, in assays completed with chromic oxide, the impact of phytase has been marginal (Selle et al., 2006; Selle and Ravindran, 2007). The position in pigs is more ambiguous. While two reports indicate that phytase substantially increases amino acid digestibility (Officer and Batterham, 1992a,b; Kornegay et al., 1998) the outcomes of the majority of swine assays are equivocal (Selle and Ravindran, 2008). However, chromic oxide was used as the marker in all the reviewed studies and as discussed by Selle et al. (2000) and Selle and Ravindran (2008), it is also possible that methods used to collect ileal digesta samples (cannulation procedures versus slaughter techniques), feeding regimen (restricted versus ad libitum feeding) and age of the test pigs (weaner versus grower– finishers) influence outcomes of phytase amino acid digestibility assays in pigs. 6.1. Underlying mechanisms Consideration is given to the possible influence of Ca on the likely mechanisms whereby phytate depresses amino acid digestibility. The likelihood is that phytate binds protein as binary protein–phytate complexes at acidic pH levels in the stomach and proventriculus of pigs and poultry (Cosgrove, 1966; Anderson, 1985). At pH levels below the isoelectric point of protein the polyanionic phytate molecule electrostatically binds with residues of the basic amino acids arginine, histidine and lysine. This initial, rapid step is followed by a progressive protein–protein aggregation that may result in precipitation (Rajendran and Prakash, 1993). The crucial proposition is that phytate-bound protein is refractory to pepsin digestion (Camus and Laporte, 1976; Knuckles et al., 1985, 1989). Camus and Laporte (1976) attributed inhibition of pepsin digestion by phytate to the formation of insoluble protein–phytate complexes under acidic conditions. Pivotally, Vaintraub and Bulmaga (1991) reported that sodium phytate reduced in vitro pepsin hydrolysis of bovine serum albumen, casein, haemoglobin and 11S soy protein by 60 to 85% at pH 2.0 but had no influence on pepsin hydrolysis at pH 4.0 to 4.5. The researchers suggested, on the basis of pepsin inhibition, that protein–phytate complexes are formed in a narrow pH range of 2 to 3. Interestingly, pepsin contains only four basic amino acid residues from a total of 327 residues per molecule (Tang et al., 1973) and this paucity should preclude any interactions between phytate and the enzyme; therefore, it is the interaction of phytate and the protein substrate that may be of importance. In this context, it is noteworthy that the peptide which activates pepsinogen contains 13 basic residues out of a total of 44 amino acids (Dunn et al., 1978), thus phytate may readily bind this activation peptide and impede the conversion of the zymogen to pepsin. Indeed, Dykes and Kay (1977) have shown that modifying lysine and histidine residues of this peptide significantly depressed activation of pepsinogen. Thus the possibility of phytate interacting with this activation peptide may be another avenue whereby phytate influences pepsin activity.

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The consequences of de novo formation of pepsinrefractory, binary protein–phytate complexes may be fundamental to the negative influence of phytate on amino acid digestibility. Complex formation could interfere with the initiation of protein digestion and the regulatory role of peptide end-products of pepsin digestion in the overall protein digestive process (Selle et al., 2000). While protein– phytate complexes dissociate once the isoelectric point of protein is exceeded, it may be that pre-bound proteins are still less readily digested in the small intestine because their solubility is reduced pursuant to their electrostatic precipitation with phytate (Lillford and Wright, 1981). While speculative, the refractory nature of complexed protein may prompt compensatory gastric hypersecretion of pepsin and hydrochloric acid (HCl). Because pepsin and HCl are ‘endogenous aggressors’ (Allen and Flemstrom, 2005), any hypersecretion would be met by additional outputs of mucus and sodium bicarbonate (NaHCO3). In high phytate diets offered to pigs, exogenous phytase has been shown to reduce gastric mucin output by 25.2%, from 7.17 to 5.36 g/day (Kies, 2005). In broilers, phytate has been shown to significantly increase mucin secretion, as assessed by sialic acid excretion, and total tract Na excretion (Cowieson et al., 2004). Also, Kies et al. (2006b) found that phytase, at inclusion rates of up 1500 FTU kg− 1, linearly increased total tract digestibility of Na by a maximum of 9.5% (0.888 versus 0.811) in pigs. Moreover in broilers, Ravindran et al. (2006) reported that phytate significantly increased Na secretion into the small intestine, which was counteracted by phytase. Conceivably, this movement of Na into the small intestine may be as NaHCO3 to buffer the phytate-induced excess of HCl. Thus, these findings are consistent with the proposal that phytate triggers gastric hypersecretion of pepsin plus HCl and, in turn, mucin. This capacity of phytate to drag Na into the small intestinal lumen, which is ameliorated by phytase, may mean that phytate is compromising intestinal uptakes of dietary and endogenous amino acids by impeding Na+dependent transport systems and Na+,K+-ATPase activity, the so-called ‘sodium pump’ (Glynn, 1993). Studies by Selle et al. (2007) and Ravindran et al. (2008) provide some support for the proposition that phytate depresses intestinal uptakes of amino acids, which is counteracted by phytase. Additionally, phytate has been shown to exacerbate endogenous amino acid flows at the ileal level in broilers in (Cowieson and Ravindran, 2007a; Cowieson et al., 2008). Clearly, phytate-induced increases in endogenous amino acid flows would stem from increased secretion and/ or decreased re-absorption of amino acids (Nyachoti et al., 2000). The protein component of mucin is approximately 350 g kg− 1 (Lien et al., 1997) so any phytate-induced outputs of pepsin and mucin would increase the secretion of endogenous amino acids, which may be compounded by their decreased re-absorption. The amino acid profiles of pepsin (Blumenfeld and Perlmann, 1958; Tang et al., 1973) and mucin (Lien et al., 1997) may be compared with the amino acid composition of the basal diet and the quantities of ileal digestible amino acids generated by phytase in broilers (Ravindran et al., 2006). The amino acid profile of pepsin was not correlated to dietary amino acids (r = 0.32; P N 0.05) but it was signi-

ficantly correlated to the amino acids generated by phytase (r = 0.61; P b 0.01). Again, mucin was not related to dietary amino acids (r = 0.17; P N 0.05) but the correlation between the amino acid profile of mucin and amino acids generated by phytase approached significance (r = 0.47; P = 0.07). These Pearson correlations are not conclusive but that amino acid profiles of pepsin and mucin are more closely correlated to the amino acids generated by phytase than the amino acid composition of the diet is consistent with the suggestion that phytate increased, and phytase reduced, endogenous amino acid flows stemming from pepsin and mucin secretions. 6.2. Possible influences of calcium The de novo formation of protein–phytate complexes in the gut, that are refractory to pepsin digestion, may be fundamental to the negative influence of phytate on amino acid digestibility. If so, the extent of complex formation is crucial and there are in vitro indications that phytate has the capacity to bind relatively large proportions of dietary protein (Kies et al., 2006a). Given that refractory protein– phytate complexes are formed in a narrow pH range of 2 to 3 (Vaintraub and Bulmaga, 1991), pH in the stomach or proventriculus is critical. Calcium will tend to increase gut pH because of the high acid binding capacity of limestone and this may reduce complex formation and mute the negative impact of phytate on protein digestibility. Pontoppidan et al. (2007) suggested that increasing Ca: phytate ratios will counteract the precipitation of protein by phytate and these workers reported that Ca modestly increased the solubility of phytate and protein between pH 2 and 5. However, Prattley et al. (1982) reported that additional Ca reduced the amount of bovine serum albumin bound to sodium phytate by approximately 40% over the same pH range. Hill and Tyler (1954b) reported that wheat gluten and sodium phytate formed insoluble protein–phytate complexes at pH 3 but the addition of limestone substantially increased the solubility of these complexes. Okubo et al. (1974a,b, 1976) investigated phytate binding of glycinin, the major globulin in soy protein, and assessed the effects of Ca on complex formation. Ca promoted phytate binding above the isoelectric point of glycinin (pH 4.9), which represents ternary protein– phytate complex formation where phytate and protein are linked by a cationic bridge. In contrast, below the isoelectric point of glycinin, Ca decreased the stability and formation of protein–phytate complexes and sufficient Ca was able to dissociate glycinin–phytate complexes at pH 3. This was attributed to Ca directly competing with basic protein residues for the negatively charged P moieties of phytate. In fact, the capacity of Ca to release protein from binary complexes at acidic pH has been adopted to prepare phytatefree soy protein isolates (Okubo et al., 1975). Essentially, these findings clearly demonstrate the potential of Ca to disrupt protein–phytate complex formation. Also, Ca may interact with soy protein, as reported by Kroll (1984) who concluded that Ca ions were bound to soy protein via side-chain carboxyl groups of aspartic acid and glutamic acid and imidazole groups of histidine. Ca–protein binding increased with pH levels from approximately 2 moles of Ca2+ per 105 g soy protein at pH 3 to a peak of 25 moles at pH 7.

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Subsequently, Gifford and Clydesdale (1990) reported that phytate reduced in vitro solubility of soy protein concentrate by 82% (26 versus 147 mg soluble protein) at pH 2.0; however, Ca also reduced soy protein solubility by 59% to (61 mg versus 147 mg). These reports indicate that Ca has the potential to bind soy protein at low pH; however, there are reports where Ca binding with 11S soy protein was only evident at alkaline pH levels of 7.8–8.0 (Appurao and Narasinga Rao, 1975; Sakakibara and Noguchi, 1977). There is the suggestion that dietary concentrations of Ca, phytate and protein may collectively influence the extent of protein–phytate complex formation in the stomach by Ca interacting with phytate or protein at acidic pH. High Ca concentrations, via these interactions and by increasing pH, may have the capacity to diminish refractory protein– phytate complex formation and, consequently, the magnitude of ileal amino acid digestibility responses to phytase supplementation. 6.3. In vivo evidence of the influence of calcium Consequently, the study by Ravindran et al. (2000) is relevant where the effects of exogenous phytase on AID of eight essential amino acids in broilers offered diets based on a wheat–sorghum blend were determined. Overall, 800 FTU kg− 1 A. niger phytase increased the AID of eight amino acids by an average of 3.75% with responses ranging from 1.06 to 7.45%. However, this experiment embraced a range of dietary concentrations of Ca (8.7 to 13.9 g kg− 1), phytate (12.06 to 22.34 g kg− 1) and protein (213 to 221 g kg− 1). It may be deduced from these analysed values that there were significant, negative correlations between phytaseinduced percentage increases in amino acid digestibility and both Ca:phytate ratios (r = −0.61; P b 0.001) and Ca:protein ratios (r = − 0.43; P b 0.01). The relevant multiple linear regression equation (R = 0.74; P b 0.001) is as follows: yðmean % phytase responseÞ = 14:999 − 10:061⁎Ca : phytate − 78:578⁎Ca : protein: Then phytase supplementation (800 FTU kg− 1) of a hypothetical broiler diet containing 10 g/kg Ca, 10 g kg− 1 phytate and 200 g/kg protein would increase amino acid digestibility by an average of 1.01% based on this equation. However, a reduction in dietary Ca content to 5 g kg− 1 would result in an 8.0% increase in amino acid digestibility whereas, 15 g kg− 1 Ca would result in a decline in amino acid digestibility to −6.0%. Thus the relationships arising from the Ravindran et al. (2000) study indicate that dietary Ca concentrations, relative to phytate and protein, could have profound effects on responses to exogenous phytase in ileal amino acid digestibility assays in broilers. 6.4. Ternary calcium–protein–phytate complexes Phytate has the capacity to bind protein, via a cationic bridge, to form ternary complexes at relatively alkaline pH where Ca usually links protein and phytate. Reddy and Salunkhe (1981) reported that binary protein–phytate complexes were formed at pH 2.8, whereas ternary complexes

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were formed at pH 8.4, via Ca, Mg and Zn cationic bridges in black gram cotyledons. Thus it is possible, as suggested by Champagne (1988), that the following equilibrium exists in the gut ½protein–Ca–phytate ⇔ protein þ Ca þ phytate ⇔ ½Ca–phytate þ protein: Thus, Ca may interact with phytate and protein in the small intestine by forming ternary protein–phytate complexes. However, Champagne et al. (1990) suggested that, as protein would be mainly present as lower molecular weight peptides, ternary complexes may not have the capacity to bind sufficient protein to be important in respect of amino acid digestibility. 6.5. Possible influence of dietary inorganic phosphorus Lei and Stahl (2000) argued that exogenous phytase efficacy is enhanced in diets containing minimal amounts of inorganic P supplements. Citing Greiner et al. (1993), they attributed this to the inhibition of phytase activity by inorganic P, the end-product of phytate hydrolysis. In an in vitro system, Mahajan and Dua (1997) reported that inorganic P (Na2HPO4) inhibited rapeseed phytase activity by up to 80.2%. While there is the possibility that inorganic P may inhibit phytase activity presumably, in vivo, this capacity would be diminished by the absorption of phytaseliberated P from the gastrointestinal tract in vivo. In a recent phytase amino acid digestibility assay broilers were offered maize–soy diets containing two levels of available P (Centeno et al., 2007). Phytase increased amino acid digestibility by an average of 12.0% at 1.4 g kg− 1 available P, but depressed digestibility by 2.1% at 2.7 g kg− 1 available P. Treatment interactions between phytase and available P were significant for all amino acids assessed with the exception of alanine. Centeno et al. (2007) suggested that additional dicalcium phosphate may have reduced phytate hydrolysis by exogenous phytase because of the inhibitory effect of inorganic P advanced by Lei and Stahl (2000). However, an alternative interpretation has been put forward. Martinez-Amezcua et al. (2006) determined the effect of exogenous phytase on ileal amino acid digestibility in broiler diets containing 400 g/kg distillers dried grains with solubles. An E. coli-derived phytase increased the AID coefficients of 12 amino acids by an average of 14.2% (0.790 versus 0.692) at 10,000 FTU kg− 1. However, the addition of approximately 2.05 g kg− 1 inorganic P (as 9.0 g kg− 1 KH2PO4) increased AID coefficients by 16.6% (0.807 versus 0.692). Martinez-Amezcua et al. (2006) considered that the comparable increases in amino acid digestibility generated by the high phytase inclusion and KH2PO4 stemmed from the provision of additional P because P is essential for transport systems involved in the intestinal uptake of amino acids. Then it is relevant that additional available P increased AID coefficients of amino acids by an average of 8.0% (0.812 versus 0.752) in the Centeno et al. (2007) study, which curiously, were not significant for any amino acid. Consequently, if basal diets are sufficiently P-inadequate then phytase-generated increases in amino acid digestibility may stem from the liberation of phytate-bound P.

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7. Calcium and the ‘energy effect’ of phytate and phytase Exogenous phytase consistently enhances energy utilisation in poultry as assessed by increased apparent metabolisable energy (AME) densities in broiler diets (Selle and Ravindran, 2007). However, this positive effect is not equally evident in terms of digestible energy in pigs (Selle and Ravindran, 2008). Also, the mechanisms whereby phytate depresses energy utilisation have not been properly identified. It has been suggested that phytate complexes with starch and reduced its digestibility (Thompson, 1988); however, very little evidence exists to support the concept of phytate–starch complexes. Numerous studies, including reports by Sharma et al. (1978) and Desphande and Cheryan (1984), have demonstrated that phytate has the capacity to inhibit α-amylase activity, in which Ca–phytate interactions may play a role, but it has not been established that phytate inhibition of α-amylase in pigs and poultry is of nutritional importance. Phytate has been shown to reduce blood glycaemic indices in humans (Thompson et al., 1987); but this may be related to depressed intestinal glucose uptakes rather than impaired starch digestion (Rickard and Thompson, 1997). To the extent that phytate depresses protein digestibility there will be a corresponding reduction in energy utilisation, which has been considered in relation to phytate-induced endogenous amino acid losses by Cowieson and Ravindran (2007b). However, phytase-induced increases in protein digestibility do not appear to be sufficient to account entirely for enhanced energy utilisation. Ravindran et al. (2000) proposed that phytate, as Ca–phytate, is involved in the formation of metallic soaps in the gut and, earlier, Cosgrove (1966) described ‘lipophytins’ as a complex of Ca/Mg– phytate, lipids and peptides. Metallic soaps are a major constraint on the utilisation of energy derived from lipids, particularly saturated fats, in broiler diets (Leeson, 1993). So it follows that phytate degradation and reduced metallic soap formation may enhance utilisation of energy derived from lipids and phytase has been shown to increase ileal fat digestibility in broilers (Camden et al., 2001). While Ca is implicated, the ‘energy effect’ of phytase requires clarification. It is possible that determining the impact of phytase on net energy, rather than digestible energy in pigs and metabolisable energy in poultry, would prove more instructive. This is illustrated by the findings of Olukosi et al. (2008). These workers reported that 1000 FTU kg− 1 phytase increased daily metabolisable energy intakes by 17.6% (734 versus 624 kJ) in broilers to 21-days of age. However, the relative increase of 23.8% in net energy intakes was greater (3284 versus 230 kJ day− 1). Thus net energy appears to be a more sensitive parameter to assess the impact of phytate and phytase on energy utilisation in broilers.

amended soils and the threat of eutrophication in fresh-water ecosystems (Leytem et al., 2007a). Therefore, it is a potential concern that phytase may be more effective in reducing total P rather than water soluble P in excreta (Miles et al., 2003). However, Leytem et al. (2006) found that concentrations of water soluble (extractable) P and phytate-P, as proportions of total P in poultry excreta, are linearly related (r2 = 0.94) in poultry excreta and both dietary Ca levels and Ca:available P ratios are critical to the water soluble P content of broiler litter (Leytem et al., 2007a). Subsequently, Leytem et al. (2007b) reported that reducing phytate levels in broiler diets from 2.8 to 1.0 g kg− 1 with low-phytate soybean meal reduced total P (62.7%), water soluble P (66.4%) and phytateP (74.6%) in excreta by broadly similar magnitudes. Also, increasing dietary Ca from 4.7 to 11.6 g kg− 1 increased the amount of phytate-P (43.0%) excreted but decreased the output of water soluble P (57.0%). In pigs, Beaulieu et al. (2007) found that E. coli phytase reduced total P and soluble P outputs, but high Ca:P ratios diminished this effect. Largely on the basis of empirical evidence, phytase feed enzymes have been associated with poor litter quality in broiler production (Debicki-Garnier and Hruby, 2003). However, it has not been established that phytases are directly responsible for ‘wet litter’ problems. Anecdotally, elevated K levels and/or increased dietary electrolyte balances (DEB) levels pursuant to the replacement of meat-and-bone meal with soyabean meal in broiler diets have been causative factors. It is relevant, therefore, that increasing DEB from 150 to 375 meq/kg− 1 has been shown to significantly downgrade excreta scores and reduce excreta dry matter from 26.9 to 18.8% (Ravindran et al., 2008). Of relevance, is that exogenous phytase had no influence on excreta scores or excreta dry matter in the same experiment. However, Murukami et al. (1997) found that increasing dietary Na levels had significant negative impacts on litter scores in broilers at 21 and 56 days of age. This suggests that the ‘Na-sparing’ effect of exogenous phytase may be relevant and diets should be adjusted accordingly. Interestingly, Pos et al. (2003) reported significant improvements in litter quality scores following reductions in Ca of 1.6 g kg− 1 in phytase-supplemented diets. In addition, this Ca reduction significantly enhanced feed intake (3.4%) and weight gain (3.1%). Without this reduction in Ca, two exogenous phytases reduced litter quality scores from 6.2 to either 5.2 or 5.3 in broilers at 28 days of age. Bedford et al. (2007b) concluded that while litter moisture may be influenced by feeding diets adjusted for phytase inclusion but that these effects may be mitigated by appropriately adjusting dietary mineral contents. Thus, the issues of water soluble P in pig and poultry excreta and the wet litter problem in broilers are further illustrations of the impact of Ca on the phytate–phytase axis in nutrition and ecology.

8. Calcium, phytate, phytase and excreta quality

9. Concluding remarks

The capacity of phytase feed enzymes to reduce P in excreta and ameliorate environmental pollution from pig and poultry production was fundamental to their development and introduction. However, from an ecological standpoint, the concentration of water soluble P relative to total P in pig and poultry excreta is critical in relation to P run-off from manure

Exogenous phytase is usually added to pig and poultry diets at approximately 500 FTU kg− 1 but, given declining inclusion costs coupled with increasing feed ingredient prices; higher phytase addition rates may be justified. Shirley and Edwards (2003) evaluated phytase at graded inclusion rates up to 12,000 FTU kg− 1 in broilers offered maize–soy

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diets containing 8.8 g kg− 1 Ca, 2.72 g kg− 1 phytate-P and 1.88 g kg− 1 nonphytate P. As shown in Table 7, responses to phytase in growth performance, mortality, P, Ca and N retention, energy utilisation and phytate-P disappearance were of a considerably greater magnitude at 12,000 FTU kg− 1 phytase activity than at conventional inclusion rates. The possibility that elevated phytase inclusion rates will be adopted in practice presents the question as to appropriate adjustments in dietary Ca and P levels and Ca:P ratios. In respect of Ca, the fundamental problem is that while in vitro data suggest that phytate binds five Ca atoms this may not apply in small intestinal digesta of pigs and poultry where cations with greater affinity for phytate are present. From the Farkvam et al. (1989) study, it may be deduced that phytate binds four Ca atoms in broiler diets containing 7.3 g kg− 1 Ca and 10.0 g kg− 1 phytate. Then, if a phytase feed enzyme was completely degraded phytate it would theoretically liberate 2.82 g kg− 1 P and 2.43 g kg− 1 Ca. The capacity of phytate to bind Ca in the small intestine needs to be established so that appropriate adjustments to dietary Ca levels in phytasesupplemented diets, particularly at elevated inclusion rates, can be made. Clearly, to facilitate phytase activity, dietary Ca levels should be kept to a minimum in phytase-supplemented pig and poultry diets but without compromising skeletal integrity or growth performance. Interestingly, Driver et al. (2005b) concluded that the NRC requirement of 10.0 g kg− 1 Ca is appropriate in starter chicks (0–3 weeks) but 9.0 g kg− 1 Ca may be excessive for growth performance and bone mineralisation in grower chicks (3–6 weeks). Recently, Bunzen et al. (2007) investigated Ca and P requirements in maize–soy diets with Ca:available P ratios of 2:1 in broilers from 22 to 35 days of age. Based on feed efficiency, these workers recommended levels of 7.54 g kg− 1 Ca and 3.77 g kg− 1 available P for male and 6.82 g kg− 1 Ca and 3.41 g kg− 1 available P for female chicks. These recommendations are substantially lower than NRC (1994) guidelines and suggest that more conservative Ca levels could be adopted; however the study would be more relevant had the diets been supplemented with phytase. In the present context, it is now necessary to define appropriate dietary nonphytate P and Ca levels for pigs and poultry in diets supplemented with microbial phytase across a range of inclusion rates. Table 7 Effects of phytase inclusion rates on broiler growth performance at 16 days of age, P and Ca retention, protein (N retention) and nitrogen-corrected apparent metabolisable energy (AMEn) utilisation and total tract phytate-P disappearance (adapted from Shirley and Edwards, 2003). Parameter

Weight gain (g bird−1) Feed intake (g bird− 1) Gain:feed (g g− 1) Mortality (%) P retention Ca retention N retention AMEn (kcal kg) Phytate-P disappearance a

Aspergillus niger phytase inclusion rates Control (0 FTU kg− 1)

Conventional (563 g kg− 1) a

High (12000 FTU kg− 1)

287 381 0.755 13.0 0.510 0.456 0.584 3216 0.403

412 498 0.827 8.5 0.573 0.432 0.705 3358 0.540

515 595 0.866 0.0 0.799 0.534 0.777 3415 0.948

Mean of 375 and 750 FTU kg− 1.

137

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