Enzymes and their effect on amino acid nutrition Mike Bedford and Carrie Walk, AB Vista Feed Ingredients, Marlborough, Wilts UK.

Whenever the application of enzymes to improve protein digestibility is discussed, one of the immediate classes of enzymes that comes to mind is protease. However, this is not the only feed enzyme that can directly influence protein digestibility. In some cases proteases may be completely overshadowed by the effects of the other two main classes of enzymes used, namely phytases and non-starch polysaccharideases (NSP’ases). A very brief explanatory review follows. Scene setting – why is protein digestion compromised? The digestion of protein is driven by the presence of endogenous proteases and in the case of the monogastric this is a two stage process. The first phase is the gastric phase which is low pH and exposes the proteins to pepsin. The second phase is the small intestinal phase, a neutral phase where trypsin, chymotrypsin, elastase and several other exo-proteases are present to complete the process of protein digestion. The secreted proteases are very effective in degrading dietary proteins and as a result are potentially dangerous as they could digest the animal’s gastrointestinal (GI) tract and the cells in which they are produced. However, this problem is averted since the enzymes are secreted in an inactive form and only activated by pH or enzymes within the lumen . In addition, the gut is protected by a layer of mucus which is relatively inert to proteolytic destruction. Generally this system works well but protein digestion may be compromised for a number of reasons including: 1. Protease inhibitors within feed ingredients 2. Damage to intestinal structure and absorptive surface area 3. Rapid transit time through the gastrointestinal tract 4. Insufficient secretion of endogenous proteases The latter includes impediments such as viscous non-starch polysaccharides (NSPs) which reduce the turnover rate of all digestive enzymes, including proteases, and thus the net result is that there may be insufficient proteases secreted to facilitate complete digestion. Young and diseased animals may also be compromised in their ability to produce or secrete digestive enzymes in which case nutrient digestion, particularly protein digestion, will be reduced. In many cases the animal is faced with one or more of the above situations and thus protein digestion is often not optimal. Under such circumstances, supplementation of the diet with enzymes which address one or more of the factors compromising digestion would enable more complete protein digestion and presumably more efficient growth. Proteases Augmentation of proteolytic activity in the GI tract clearly should be of advantage when digestion is overwhelmed. The proteases used commercially to date tend to be serine proteases isolated from Bacillus spp which maintain optimal activity in the alkaline region of the GI tract, in some cases their optimum activity is as high as pH 9, which is much higher than any pH encountered within the gut lumen. This suggests that they will be most active in the small intestine and likely not active in the gastric phase at all. The data to date are not consistent and perhaps this relates to

variation in response which is governed by ingredients used, the presence of other enzymes and the age of the animal. Each is discussed briefly below. Recent work has shown significant improvements in amino acid digestibility when proteases are employed but the concomitant improvement in performance is not always apparent (Angel et al., 2011; Liu et al., 2013). Several authors have suggested that the benefit derives from the ability of the exogenous proteases to target protease inhibitors and/or lectins. However, in the work of Liu et al (2013), it was suggested that, given the pattern of effect on digestibility of specific amino acids, the enzyme was probably preferentially targeting the cereal portion of the ration (Liu et al., 2013). Variation in performance of proteases depending upon dietary formulation has also been observed suggesting that the efficacy of a protease may be dependent upon the ingredients used in the ration (Kocher et al., 2003). The adjustments made to a ration when employing a protease may therefore need to take into account the ingredients used. The benefit of a protease may also depend upon the presence of other enzymes, as it has been shown that the benefit is lost when it is tested in the background of a xylanase and/or phytase (Kalmendal, 2012; Sultan et al., 2011), although addition of a protease into a ration which already contained a xylanase and an amylase was shown to improve apparent ileal amino acid digestibility and AMEn in young broilers (Romero et al., 2013). The discussion regarding how matrices of enzymes combine when different classes of enzymes are used in the same ration has led to much debate but the general conclusion is that they are not additive (Cowieson and Bedford, 2009a) and is discussed briefly below. Regardless, the implementation of a protease in a ration requires formulation advice which results in no loss in performance, and this has been the case in some (Angel et al., 2011; Yan et al., 2012) but not all performance trials reported (Tempra, 2013). In the work of Yan et al (2012) it was clear that the benefit of the protease was greater in the starter phase compared with the finisher phase which suggested that the young animal may be more responsive to such products. If correct then age specific recommendations would refine the protease offering considerably.

NSP EnzymesThe ability of an NSP’ase to enhance protein digestibility is well documented (Bedford et al., 1998; Danicke et al., 1997; Silva et al., 1997) and is driven by one of three mechanisms – reduced intestinal viscosity, direct endosperm cell wall puncturing and thus exposure of contents to digestion, and production of fermentable oligosaccharides. The viscosity mechanism is more relevant for small grain cereals such as wheat and barley, but its effect cannot be discarded for maize based diets, particularly with reference to fat digestibility. The likelihood that the third mechanism is actually responsible for increased cell wall puncturing has recently been discussed (Masey O'Neill et al., 2012; Singh, 2012). In essence it is suggested that the NSP’ase provides oligosaccharides which are fermented to volatile fatty acids (VFAs) in the large intestine in such concentrations that they trigger an entero-hormonal response which results in delayed gastric emptying (Goodlad et al., 1987) and duodenal transit rates (Park et al., 2013). This encourages more complete gastric and proximal small intestinal digestion of the whole diet, which fits with the data in the literature. Indeed when NSP’ases are used the digestibility of all amino acids in the undigested fraction is improved by approximately 15-17% (Cowieson and Bedford, 2009a; Cowieson and Bedford, 2009b), suggesting the enzyme is not just targeting the cereal fraction. If NSP’ases were specifically targeting the cell walls of the cereal fraction then it would be expected that the benefit would be greatest for those amino acids which are dominant in the cereal and this clearly is not the

case. One consequence of this proposed general tenet is that the goal for NSP’ase enzymes should be to provide the correct oligosaccharides to propel the gastric/duodenal response. Thus the search for more and more ancillary NSP degrading enzymes to “digest” the fibre fraction is likely a strategy which will ultimately fail to improve performance as the complete dissolution of cereal cell walls is clearly not responsible for the response to NSP’ases in monogastrics. Indeed the fact the effect of an NSP’ase is is generalised across the whole diet lends credence to the oligosaccharide mechanism whilst challenging that of the cell wall mechanism. Phytases A great deal of literature has focussed on the topic of amino acid digestibility and the effect of phytases, with many authors showing significant benefits (Agbede et al., 2010; Augspurger and Baker, 2004; Cadogan et al., 2009; Onyango et al., 2004; Onyango et al., 2005; Pirgozliev et al., 2011; Ravindran et al., 1998; Ravindran et al., 1999; Ravindran et al., 2001; Selle et al., 2003). However, several papers have shown no benefit of phytase on this parameter (Agbede et al., 2010; Augspurger and Baker, 2004; Boling-Frankenbach et al., 2001; Peter and Baker, 2001; Peter et al., 2000), although Boling-Frankenbach et al (2001) did suggest the effects of phytase were ingredient specific. The current understanding is that phytate negatively interferes with gastric digestion and the concomitant GIT response, i.e. increased pepsin and HCl and mucin production, is responsible for the apparent reduction in digestibility of amino acids (Cowieson et al., 2004; Cowieson et al., 2006; Cowieson et al., 2009; Cowieson et al., 2011; Peter and Baker, 2001; Peter et al., 2000). Thus the application of a phytase under the right circumstances will reduce endogenous inputs for protein digestion which results in an improvement in digestibility of those amino acids which predominate in the endogenous losses, specifically cysteine, aspartate, threonine, glycine and serine (Cowieson et al., 2004; Cowieson et al., 2006; Peter and Baker, 2001; Peter et al., 2000; Selle et al., 2012). The fact that phytate varies in its ability to interfere with gastric digestion depending upon the identity of the dietary protein investigated may explain some of the inconsistency in the literature (Kies, 2006). Generally the use of a phytase can be associated with a reduced endogenous input rather than an improvement in digestibility of the diet per se, and as a result the amino acid matrices applied to the enzyme should reflect this fact. Thus lysine and methionine should not feature as significant amino acids in the matrix of any phytase.

Combinations of enzymes This topic has been dealt with elsewhere in great detail but the basic tenet is that the phytase will reduce endogenous losses as a result of its ability to facilitate gastric and subsequent small intestinal digestion (Cowieson and Bedford, 2009a; Cowieson and Bedford, 2009b). Specific amino acids are spared more so than others when a phytase is employed, such as those involved in mucin and endogenous secretions. If an NSP’ase is then utilised it will recover approximately ~15% of the remaining undigested fraction, which will clearly include the endogenous inputs. Thus when a phytase is used in addition to an NSP’ase the amino acid matrix of the NSP’ase needs to be adjusted downwards to take into account the reduction in the undigested fraction of those amino acids recovered by the phytase. The tertiary combination with a protease now needs to take into account that the combination of both the phytase and NSP’ase has reduced endogenous losses and recovered 15% of the “undigested” fraction, the latter probably being the most accessible amino acids which escaped digestion in the absence of the NSP’ase. Application of a matrix for amino acids

for a protease derived from diets which did not contain a phytase or a xylanase therefore needs to be considered with caution. In fact, Sultan et al (2011) suggest that the combination of more than two enzyme classes from phytase, protease and xylanase, did not result in any further improvement in N digestibility. Each enzyme alone or in combination with one other tended to improve N digestibility, but addition of the third to a combination of the two resulted in no further improvement. Users need to be aware of such limitations when applying feed enzymes as the values on paper look attractive, but the biological value is less attractive and may result in significant losses in performance as the enzymes fail to deliver their matrices in an additive manner.

Reference List Agbede, J.O., Kluth, H., Rodehutscord, M., 2010. Studies on the effects of microbial phytase on amino acid digestibility and energy metabolisability in caecectomised laying hens and the interaction with the dietary phosphorus level. Br. 50, 583-591. Angel, C.R., Saylor, W.W., Vieira, S.L., Ward, N.E., 2011. Effects of a monocomponent protease on performance and protein utilisation in 7-22 day-old broiler chickens. Poult. Sci 90, 22812286. Augspurger, N.R., Baker, D.H., 2004. High dietary phytase levels maximize phytate-phosphorus utilization but do not affect protein utilization in chicks fed phosphorus- or amino aciddeficient diets. Journal of Animal. Science 82, 1100-1107. Bedford, M.R., Scott, T.A., Silversides, F.G., Classen, H.L., Swift, M.L., Pack, M., 1998. The effect of wheat cultivar, growing environment and enzyme supplementation on digestibility of amino acids by broilers. Can. J. Anim. Sci. 78, 335-342. Boling-Frankenbach, S.D., Peter, C.M., Douglas, M.W., Snow, J.L., Parsons, C.M., Baker, D.H., 2001. Efficacy of phytase for increasing protein efficiency ratio values of feed ingredients. Poult. Sci 80, 1578-1584. Cadogan, D.J., Selle, P.H., Partridge, G.G., Ravindran, V., 2009. Supplementation of wheat-based broiler diets with xylanase and phytase, individually and in combination. In: DANISCO animal nutrition seminar (Ed.). Cowieson, A.J., Acamovic, T., Bedford, M.R., 2004. The effects of phytase and phytic acid on the loss of endogenous amino acids and minerals from broiler chickens. In: pp. 101-108. Cowieson, A.J., Acamovic, T., Bedford, M.R., 2006. Phytic acid and Phytase: Implications for Protein Utilization by Poultry. Poult. Sci 85, 878-885. Cowieson, A.J., Bedford, M.R., 2009a. The effect of phytase and carbohydrase on ileal amino acid digestibility in monogastric diets: complimentary mode of action? Worlds Poultry Science Journal 65, 609-624. Cowieson, A.J., Bedford, M.R., 2009b. The law of dinishing returns: consequences for feed enzyme strategy. In: pp. 16-18.

Cowieson, A.J., Bedford, M.R., Selle, P.H., Ravindran, V., 2009. Phytate and microbial phytase:implications for endogenous nitrogen losses and nutrien availability. Worlds Poultry Science 65, 401-417. Cowieson, A.J., Wilcock, P., Bedford, M.R., 2011. Superdosing effects of phytase in poultry and other monogastrics. Worlds Poultry Science Journal 67, 225-235. Danicke, S., Simon, O., Jeroch, H., Bedford, M.R., 1997. Interactions between dietary fat type and xylanase supplementation when rye-based diets are fed to broiler chickens. 2. Performance, nutrient digestibility and the fat-soluble vitamin status of livers. Br. 38, 546-556. Goodlad, R.A., Lenton, W., Ghatei, M.A., Adrian, T.E., Bloom, S.R., Wright, N.A., 1987. Proliferative effects of fibre on the intestinal epithelium:relationship to gastrin. enteroglucagon and PYY. Gut 28, 221-226. Kalmendal, R., 2012. Effects of a xylanase and protease, individually or in combination, and an ionophore coccidiostat on performance, nutrient utilization, and intestinal morphology in broiler chickens fed a wheat-soybean meal-based diet. Kies, A.K., 2006. Interaction between protein, phytate and microbial phytase. In vitro studies. Kocher, A., Choct, M., Ross, G., Broz, J., Chung, T.K., 2003. Effects of enzyme combinations on apparent metabolizable energy of corn-soybean meal-based diets in broilers. In: pp. 275283. Liu, S.Y., Selle, P.H., Court, S.G., Cowieson, A.J., 2013. Protease supplementation of sorghum-based diets enhances amino acid digestibility coefficients in four small intestinal sites and accelerates their rates of digestion. In. Masey O'Neill, H.V., Haldar, S., Bedford, M.R., 2012. The role of peptide YY in the mode of action of dietary xylanase. Poultry Science Abstracts. Onyango, E.M., Bedford, M.R., Adeola, O., 2004. Efficacy of an Escherichia coli phytase on growth performance, nutrient utilization and bone characteristics in broiler chicks. Poult. Sci 83, 149. Onyango, E.M., Bedford, M.R., Adeola, O., 2005. Efficacy of an evolved Escherichia coli phytase in diets of broiler chicks. Poult. Sci 84, 248-255. Park, J.H., Kwon, O.K., Ahn, S.H., Lee, S., Choi, B.K., Jung, K.Y., 2013. Fatty diets retarded the propulsive function of and attenuated motility in the gastrointestinal tract of rats. Nutrition Research 33, 228-234. Peter, C.M., Baker, D.H., 2001. Microbial phytase does not improve protein-amino acid utilization in soybean meal fed to young chickens. Journal of Nutrition 131, 1792-1797. Peter, C.M., Han, Y., Boling-Frankenbach, S.D., Parsons, C.M., Baker, D.H., 2000. Limiting order of amino acids and the effects of phytase on protein quality in corn gluten meal fed to young chicks. J. An. Sci. 78, 2150-2156. Pirgozliev, V., Bedford, M.R., Acamovic, T., Mares, P., Allymehr, M., 2011. The effects of supplementary bacterial phytase on dietary energy and total tract amino acid digestibility when fed to young chickens. Br. 52, 245-254.

Ravindran, V., Bryden, W.L., Cabahug, S., Selle, P.H., 1998. Impact of microbial phytase on the digestibility of protein, amino acids and energy in broilers. Proc. Maryland Nutrition Conf. for Feed Manufacturers March 12-13, 156-165. Ravindran, V., Cabahug, S., Ravindran, G., Bryden, W.L., 1999. Influence of microbial phytase on apparent ileal amino acid digestibility of feedstuffs for broilers. Poult. Sci 78, 699-706. Ravindran, V., Selle, P.H., Ravindran, G., Morel, P.C.H., Kies, A.K., Bryden, W.L., 2001. Microbial phytase improves performance, apparent metabolizable energy, and ileal amino acid digestibility of broilers fed a lysine-deficient diet. Poult. Sci 80, 338-344. Romero, L.F., Parsons, C.M., Utterback, P.L., Plumstead, P.W., Ravindran, V., 2013. Comparative effect of dietary carbohydrases without or with protease on the ileal digestibility of energy and amino acids and AME in young broilers. Animal Feed Science and Technology 181, 3544. Selle, P.H., Bryden, W.L., Pittolo, P.H., Ravindran, V., 2003. Effects of phytase supplementation of diets with two tiers of nutrient specifications on growth performance and protein efficiency ratios of broiler chickens. Asian-Australasian Journal of Animal Sciences 16, 1158-1164. Selle, P.H., Cowieson, A.J., Cowieson, N.P., Ravindran, V., 2012. Protein-phytate interactions in pig and poultry nutrition: a reappraisal. Nutr. 25, 1-17. Silva, S.S.P., Gilbert, H.J., Smithard, R.R., 1997. Exogenous polysaccharides do not improve digestion of fat and protein by increasing trypsin or lipase activities in the small intestine. Br. 38, S39S40. Singh, A., 2012. Effects of xylanase supplementation on performance, total volatile fatty acids and selected bacterial populations in caeca, metabolic indices and peptide YY concentrations in serum of broiler chickens fed energy restricted maize-soybean based diets. Sultan, A., Gan, C.Y., Li, X., Zhang, D., Bryden, W.L., 2011. Dietary enzyme combinations improve sorghum ileal protein and starch digestibility during the broiler starter phase. In: p. 82. Tempra, M.A., 2013. Proteases for broilers and layers: do they work? Asian Poultry 2013 July : pp. 34-35. Yan, F., Garribay, L., Arce, J., Lopez-Coello, C., Camacho, D., Disbennet, P., Vazquez-Anon, M., Manangi, M., Odetallah, N.H., Carter, S., 2012. Effects of protease supplementation on broiler performance and in vitro protein digestibility. Australian Poultry Science Symposium, 134-137.