A. J. Cowieson

THE EFFECTS OF PHYTASE AND PHYTIC ACID ON THE LOSS OF ENDOGENOUS AMINO ACIDS AND MINERALS FROM BROILER CHICKENS

1. The effects of myo-inositol hexaphosphate (IP6) and phytase (EC 3.1.3.26) on the excretion of endogenous compounds were investigated using growing broiler chickens. 2. A total of 32 female Ross broilers were used in a precision feeding assay involving a 2×2 factorial arrangement of treatments. The materials administered were glucose, glucose + 1000 units of phytase activity (FTU), glucose + 1 g of IP6 and glucose + 1 g of IP6 + 1000 FTU. Excreta were collected quantitatively over a 48-h period following intubation of the test materials. The excretion of nitrogen, amino acids, minerals, sialic acid and phytate phosphorus was determined. 3. The ingestion of 1 g of IP6 by broilers increased the excretion of endogenous nitrogen, amino acids, iron, sodium, sulphur and sialic acid compared with birds fed on glucose. Supplementation of IP6 with exogenous phytase reduced the excretion of endogenous amino acids, calcium, sodium, phytate phosphorus and sialic acid compared with birds fed IP6. 4. It can be concluded that IP6 increases the excretion of endogenous minerals and amino acids in broiler chickens. Part of the beneficial effects of the addition of exogenous phytases to the diets of poultry appears to be mediated through a reduction in endogenous losses of these nutrients.

BOARD-INVITED REVIEW: opportunities and challenges in using exogenous enzymes to improve nonruminant animal production.

Diets fed to nonruminant animals are composed mainly of feed ingredients of plant origin. A variety of antinutritional factors such as phytin, nonstarch polysaccharides, and protease inhibitors may be present in these feed ingredients, which could limit nutrients that may be utilized by animals fed such diets. The primary nutrient utilization-limiting effect of phytin arises from the binding of 6 phosphate groups, thus making the P unavailable to the animal. The negative charges allow for formation of insoluble phytin-metal complexes with many divalent cations. Furthermore, phytin and protein can form binary complexes through electrostatic links of its charged phosphate groups with either the free amino group on AA on proteins or via formation of ternary complexes of phytin, Ca(2+), and protein. The form and extent of de novo formation of binary and ternary complexes of phytin and protein are likely to be important variables that influence the effectiveness of nutrient hydrolysis in plant-based diets. Nonstarch polysacharides reduce effective energy and nutrient utilization by nonruminant animals because of a lack of the enzymes needed for breaking down the complex cell wall structure that encapsulate other nutrients. Enzymes are used in nonruminant animal production to promote growth and efficiency of nutrient utilization and reduce nutrient excretion. The enzymes used include those that target phytin and nonstarch polysaccharides. Phytase improves growth and enhances P utilization, but positive effects on other nutrients are not always observed. Nonstarch polysaccharide-hydrolyzing enzymes are less consistent in their effects on growth and nutrient utilization, although they show promise and it is imperative to closely match both types and amounts of nonstarch polysaccharides with appropriate enzyme for beneficial effects. When used together with phytase, nonstarch polysaccharide-hydrolyzing enzymes may increase the accessibility of phytase to phytin encapsulated in cell walls. The future of enzymes in nonruminant animal production is promising and will likely include an understanding of the role of enzyme supplementation in promoting health as well as how enzymes may modulate gene functions. This review is an attempt to summarize current thinking in this area, provide some clarity in nomenclature and mechanisms, and suggest opportunities for expanded exploitation of this unique biotechnology.

Effect of exogenous enzymes in maize-based diets varying in nutrient density for young broilers: growth performance and digestibility of energy, minerals and amino acids.

1. A total of 192 male broilers (Cobb 500) were used in a growth and digestibility assay, involving a 2 × 2 factorial arrangement of treatments, to assess the effects of an enzyme cocktail of xylanase, amylase and protease in maize-based diets. 2. The following two diets were formulated: a positive control diet containing adequate nutrient concentrations for broiler starters as per breeder recommendations and a negative control diet to contain approximately 0·63 MJ/kg apparent metabolisable energy (AME) and 3% amino acids less than the positive control. 3. A further two dietary treatments were developed by supplementing each control diet with an enzyme product containing xylanase, amylase and protease. 4. Birds fed on the negative control diet had poorer weight gain and feed efficiency than those given the positive control diet. There was no effect of diet or enzyme on feed intake. The digestibility of nitrogen, calcium, phosphorus and most amino acids were unaffected by dietary nutrient density. 5. Supplementation of both the positive and negative control diets with the enzyme improved weight gain and feed efficiency compared with the unsupplemented diets. In the case of the negative control, supplemental enzyme improved performance to that of the unsupplemented positive control diet. There was no interaction between diet and enzyme for either weight gain or FCR, suggesting similar beneficial responses regardless of the nutrient density of the diet. 6. In both diets, enzyme supplementation improved AME by an average of 3% (0·35 MJ/kg DM) and nitrogen retention by an average of 11·7% (26 g/kg DM intake vs 29 g/kg DM intake). There was no interaction between diet and enzyme for AME or nitrogen retention. 7. Ileal digestibilities of calcium and phosphorus were not influenced by supplemental enzyme, whereas the digestibility of nitrogen and most amino acids was improved by enzyme addition compared with the unsupplemented control diets. There was no interaction between diet and enzyme for the ileal digestibility of nitrogen and amino acids. 8. These data demonstrate that it is possible to improve the nutritional value of a maize/soy-based diet for broiler starters through the use of exogenous enzymes. The nutrient density of the diet does not appear markedly to influence the response to enzyme, offering flexibility in the use of enzymes for maize-based diets. 9. It is concluded that the energy and amino acid values of maize-based diets for broilers can be enhanced by supplementation with an enzyme cocktail of xylanase, amylase and protease, offering potential economic benefits to producers.

Protein–phytate interactions in pig and poultry nutrition: a reappraisal

Protein-phytate interactions are fundamental to the detrimental impact of phytate on protein/amino acid availability. The inclusion of exogenous phytase in pig and poultry diets degrades phytate to more innocuous esters and attenuates these negative influences. The objective of the present review is to reappraise the underlying mechanisms of these interactions and reassess their implications in pig and poultry nutrition. Protein digestion appears to be impeded by phytate in the following manner. Binary protein-phytate complexes are formed at pH levels less than the isoelectric point of proteins and complexed proteins are refractory to pepsin digestion. Once the protein isoelectric points are exceeded binary complexes dissociate; however, the isoelectric point of proteins in cereal grains may be sufficiently high to permit these complexes to persist in the small intestine. Ternary protein-phytate complexes are formed at pH levels above the isoelectric point of proteins where a cationic bridge links the protein and phytate moieties. The molecular weights of protein and polypeptides in small-intestinal digesta may be sufficient to allow phytate to bind nutritionally important amounts of protein in ternary complexes. Thus binary and ternary complexes may impede protein digestion and amino acid absorption in the small intestine. Alternatively, phytate may interact with protein indirectly. Myo-inositol hexaphosphate possesses six phosphate anionic moieties (HPO(4)(2-)) that have strong kosmotropic effects and can stabilise proteins by interacting with the surrounding water medium. Phytate increases mucin secretions into the gut, which increases endogenous amino acid flows as the protein component of mucin remains largely undigested. Phytate promotes the transition of Na(+) into the small-intestinal lumen and this suggests that phytate may interfere with glucose and amino acid absorption by compromising Na(+)-dependent transport systems and the activity of the Na pump (Na(+)-K(+)-ATPase). Starch digestion may be depressed by phytate interacting with proteins that are closely associated with starch in the endosperm of cereal grains. While elucidation is required, the impacts of dietary phytate and exogenous phytase on the site, rate and synchrony of glucose and amino acid intestinal uptakes may be of importance to efficient protein deposition. Somewhat paradoxically, the responses to phytase in the majority of amino acid digestibility assays in pigs and poultry are equivocal. A brief consideration of the probable reasons for these inconclusive outcomes is included in this reappraisal.

Super-dosing effects of phytase in poultry and other monogastrics

Phytases have been used commercially since the early 1990s and have been the focus of considerable and sustained research for many decades. Despite this heroic effort there are still areas of persistent uncertainty such as the obscurity surrounding total compared with digestible calcium, appropriate modification to dietary sodium (and other electrolyte) concentrations, the usefulness of the amino acid and energy digestibility improvements and ultimately the effect of phytase on nutrient requirement. One further area which has attracted some attention recently is the effect of unconventionally high doses of phytase (i.e. >2,500 FTU/kg from Aspergillus niger or Escherichia coli) in an attempt to ostensibly ‘de-phytinise’ the diet. The effects of such ‘super’ doses of phytase can be considerable, and often beyond that which may be reasonably expected based on improvement in P digestibility per se. This review article addresses these effects and suggests mechanisms by which they may be explained.

Effect of phytic acid and microbial phytase on the flow and amino acid composition of endogenous protein at the terminal ileum of growing broiler chickens

The effects of phytic acid and microbial phytase on the flow and composition of endogenous protein at the terminal ileum of broiler chickens were investigated using the peptide alimentation method. Phytic acid (fed as the sodium salt) was included in a synthetic diet at 8.5, 11.5 and 14.5 g/kg (or 2.4, 3.2 and 4.0 g/kg phytate-phosphorus) and each diet was fed without or with an Escherichia coli-derived microbial phytase at 500 phytase units/kg diet. A control containing no phytate was fed as a comparison to estimate basal endogenous flows. Ingestion of phytic acid increased (P < 0.05) the flow of endogenous amino acids and N by an average of 47 % at the lowest phytic acid concentration and 87 % at the highest. The addition of microbial phytase reduced (P < 0.05) the inimical effects of phytic acid on endogenous amino acid flow at all dietary phytic acid levels. The composition of endogenous protein was also influenced (P < 0.10-0.001) by increasing phytic acid concentrations and phytase addition. The effects of phytic acid and phytase on endogenous flow and composition of endogenous protein, however, varied depending on the amino acid. It is concluded that the effects of phytase on amino acid digestibility may be mediated, in part, through a route of reduced endogenous loss.

Influence of Dietary Electrolyte Balance and Microbial Phytase on Growth Performance, Nutrient Utilization, and Excreta Quality of Broiler Chickens

The possible interaction between dietary electrolyte balance (DEB=Na+K-Cl, mEq/kg of diet) and microbial phytase on the performance and nutrient utilization of broiler starters and litter quality was examined in this study. A 4 x 2 factorial arrangement of treatments was used with 4 levels of DEB (150, 225, 300, and 375 mEq/kg of diet) and 2 levels of phytase (0 and 500 phytase units/kg of diet). Experimental diets were based on corn, soybean meal, and canola meal and were formulated to contain a nonphytate P level of 3 g/kg. The DEB levels were altered by the use of sodium bicarbonate and ammonium chloride. Each diet was offered to 6 replicates of 8 birds each from d 1 to 21. Increasing the DEB values from 150 to 300 mEq/kg had no effect (P>0.05) on the weight gains and feed per gain, but the gains were lowered (P<0.05) and the feed per gain was increased (P<0.05) at 375 mEq/kg. Feed intake was unaffected (P>0.05) by DEB levels. Supplemental phytase improved (P<0.05) the weight gains and feed intake at all DEB levels. Feed per gain was lowered (P<0.05) by phytase addition, but a tendency for a DEB x phytate interaction (P=0.06) was also observed, indicating that the responses to phytase may be affected by DEB level. The responses in feed per gain were greater at the lowest DEB level, and phytase addition had no effect on feed per gain at the highest DEB level. Dietary electrolyte balance levels had no effect on the AME(n) and ileal N digestibility to 300 mEq/kg, but lowered (P<0.05) both criteria at 375 mEq/kg. Phytase addition improved (P<0.05) the AME(n) and ileal N digestibility. The improvements in AME(n) with 500 U/kg of phytase addition in 150, 225, and 275 mEq/kg DEB were 53, 60, and 38 kcal/kg of DM, respectively. The main effect of DEB was significant (P<0.05) only for the ileal availability of Na and Cl, whereas added phytase influenced (P<0.05) the ileal availability of Ca, P, Na, K, and Cl. The effects of DEB were significant (P<0.05) for apparent ileal digestibility of all amino acids, except Ala (P=0.09), Arg, Met, and cystine. In general, the digestibilities of amino acids were unaffected when the DEB level was increased from 150 to 225 mEq/kg of diet, but decreased at the 300 and 375 mEq/kg levels. Phytase addition improved (P<0.06 to 0.05) ileal digestibility of all amino acids, except Met and Tyr. Increasing DEB had adverse effects on excreta scores and DM content. Phytase addition, however, had no effect on excreta quality. The overall results of the present study suggest that variability in phytase responses in nutrient utilization may be explained, in part, by differences in dietary electrolyte levels.

The effect of phytase and carbohydrase on ileal amino acid digestibility in monogastric diets: complimentary mode of action?

As the simultaneous use of carbohydrases and phytases gains momentum it is imperative that formulators understand the magnitude of additivity of effect to allow for appropriate modification to diet nutrient balance. Though carbohydrases and phytases are often thought of as pronutrients with energy, calcium and phosphorus value, within the scientific literature there are dozens of papers on the effect of these enzymes on ileal amino acid digestibility coefficients. The effect of enzymes on ileal amino acid digestibility is instructive as patterns of response allow speculation as to mode of action and likely additivity of admixtures. A review of the scientific literature has revealed that whilst xylanases and phytases may be considered to be broadly additive in effect, on an individual amino acid basis this effect ranges from sub-additive (e.g. threonine) to synergistic (e.g. arginine). Importantly, the mean response to both xylanase and phytase for ileal amino acid digestibility can be predicted (R2=0.65 and 0.56 respectively) by polynomial equations based only on the nutritional value of the control diet. The fact that control diets with an inherently high digestibility respond poorly to enzymes explains why the use of a second enzyme will likely yield a lesser response when used on top of another, since the former has already improved digestibility characteristics. The implications of these responses, as well as suggested mechanisms of action, are discussed within practical diet formulation constraints.

Effect of diet containing phytate and phytase on the activity and messenger ribonucleic acid expression of carbohydrase and transporter in chickens.

The effect of dietary phytate and phytase on carbohydrase activity and hexose transport was investigated in broiler chickens. Diets containing phytate P (2.2 or 4.4 g/kg) with different phytase dose rates (0, 500, or 1,000 phytase units/kg) were fed to 504 female Cobb chicks for 3 wk. Diets containing high phytate concentrations depressed (P < 0.05) BW and G:F, whereas phytase supplementation improved (P < 0.05) the performance of birds. In the duodenum, phytate decreased (P < 0.05) the activities of disaccharidases, Na(+)K(+)-ATPase, and glucose concentrations by 5 to 11%, but phytase enhanced (P < 0.05) the concentrations of amylase, sucrase, maltase, Na(+)K(+)-ATPase, and glucose by 5 to 30%. In the jejunum, phytate decreased (P < 0.05) the concentrations of amylase, sucrase, Na(+)K(+)-ATPase, and glucose by 10 to 22%, and phytase alleviated the negative effect of phytate on the above variables. Ingestion of diets containing phytate also decreased (P < 0.05) serum amylase activity and glucose concentration, and phytase enhanced (P < 0.05) serum concentrations of amylase, sucrase, maltase, Na(+)K(+)-ATPase, and glucose. There were also interactions (P < 0.05) between phytate and phytase on the concentrations of serum amylase, duodenal amylase, sucrase, and jejunal glucose. Enzymatic analysis at a molecular level showed that neither phytate nor phytase influenced the mRNA expression of sucrase-isomaltase in the small intestine. Also, the investigation into the sodium glucose cotransporter gene may challenge the mechanism by which phytate interferes with glucose utilization, as partly indicated by bird performance, and transmembrane transport because diets containing increased phytate upregulated (P < 0.05) the mRNA expression of the sodium glucose cotransporter gene in duodenum and did not influence it in the jejunum. These results indicate that phytate can impair endogenous carbohydrase activity and digestive competence, and phytase can ameliorate these effects for chickens.

Intestinal function and gut microflora of broiler chickens as influenced by cereal grains and microbial enzyme supplementation

A study was conducted to investigate the effect of the key cereal grains and a microbial enzyme supplement on broiler chicken performance, gut microflora and intestinal function. Ingestion of the barley-based diet was associated with low 28-day body weight, decreased feed intake and high FCR. The supplemental enzyme increased feed intake and weight gain of the chickens on a wheat-based diet. The pH of the gizzard and caecal contents varied with the grain type. Enzyme supplementation reduced ileal viscosity, particularly in birds that received the diet based on wheat. The birds on the barley-based diet had lower ileal digestibility of dry matter, protein and energy than those given maize and sorghum-based diets. The ileal digestibility of starch was increased by enzyme supplementation. Enzyme supplementation increased the number of total anaerobic bacteria in the gizzard of birds fed on sorghum and increased lactobacilli in the gizzard of those fed both sorghum and wheat. The birds fed the sorghum-based diet had the lowest counts of caecal total anaerobic bacteria and lactobacilli. Jejunal villus height and villus:crypt ratio of birds fed the barley-based diet were the lowest when compared with those fed the other diets. Enzyme application induced an increase in villus height and villus:crypt ratio of birds on wheat, crypt depth on barley and a reduction in crypt depth of chickens on the sorghum-based diets. The highest activity of maltase and the lowest activity of sucrase were observed in tissue from birds fed on maize and sorghum-based diets respectively. The differences in the performance of broilers on cereal grains could be explained by changes in intestinal morphology, enzyme activities and gut microflora as well as nutrient digestibility. The improved performance by supplemental enzyme in wheat-fed chickens was associated with beneficial changes in intestinal morphology and digesta viscosity.