The use of exogenous proteases in poultry feed has become more prevalent in recent years, following the broader commercial acceptance of other feed enzymes like phytases and xylanases, and increased pressure on the cost of proteinaceous ingredients. Most current commercial proteases for animal feed are alkaline proteases of bacterial origin. Feed cost is reduced with the inclusion of proteases through a reduction of crude protein and first limiting amino acids supplied by dietary ingredients. Nonetheless, the effects of exogenous proteases on animal performance do not necessarily reflect the in vitro digestibility of protein from ingredients, but are influenced by a variety of factors that affect their bio-efficacy. For example, serine proteases in broiler chickens do not appear to show a linear dose response on protein digestibility of complete feeds, but an optimum is present (Argüelles-Ramos et al., 2010), after which marginal reductions with increasing protease doses are evident. This suggests that a balance between the hydrolysis of dietary protein and undefined physiological interactions in the intestine may limit further improvements in protein retention due to protease activity. Effects of exogenous proteases are not only confined to the digestion of protein, but extend to the digestion of other nutrients such as fat and starch. Furthermore, the application of proteases cannot be considered in isolation because it often occurs in combination with other feed enzymes, whose mechanisms and subsequent effects on digested nutrients do not appear to be totally independent to those of proteases.
This paper describes factors that influence the bio-efficacy and application of proteases in broiler diets with emphasis on nutrient digestibility, the interaction of proteases with other dietary enzymes, and nutritional effects of exogenous proteases beside protein digestion.
II. EFFECTS OF PROTEASES ON AMINO ACID DIGESTIBILITY
A correct estimation of an amino acid matrix to be assigned to exogenous proteases during the formulation of diets is essential to capturing their value in terms of animal performance. That is because an overestimation of the protease effect on the digestibility of essential amino acids, in particular, would create limits to the efficient utilisation of potential improvements on crude protein digestibility. A common mistake in the application of proteases is to assume that a set amount of improvement of protein digestibility, e.g. 3%, can be extrapolated for all amino acids, to calculate an amino acid matrix. In reality, individual dietary amino acids differ widely in their digestibility across different ingredients and diet types. The effect of protease will always be limited by the amount of undigested amino acids present in the small intestine in the absence of the additive. In a recent review, Cowieson (2010) concluded that the inherent digestibility of nutrients in poultry diets prior to enzyme addition is a good indicator of the magnitude of enzyme response.
Romero et al. (2009) conducted a series of studies to determine the relationship between the amount of ileal undigested amino acids and the effects of protease on the ileal digestibility of each amino acid. Four different 21-day digestibility trials were conducted. Each study evaluated the energy and amino acid digestibility of broilers fed corn-soy diets supplemented with a multi-enzyme complex containing xylanase from T. reesei, amylase from B. amyloliquefaciens, and protease from B. subtilis (Avizyme 1502; Danisco Animal Nutrition, DuPont Industrial Biosciences), compared to one containing only xylanase from T. reesei and amylase from B. amyloliquefaciens, and an un-supplemented control treatment. Diets were corn-soybean meal based in two of the trials, and additionally contained 7-10% corn DDGS in the other two trials.
Figure 1 - Percentage change of ileal digestibility of nitrogen and amino acids on a control diet with addition of two different enzyme combinations of carbohydrases with (XAP) or without (XA) a serine protease in broiler chickens.
On average across all four studies, the addition of the xylanase/amylase combination to the un-supplemented control diet increased ileal digestible energy by 78 kcal/kg, whereas the xylanase/amylase/protease combination increased it by 100 kcal/kg. Most notably, the xylanase/amylase/protease treatment resulted in significantly higher digestibility of nitrogen and all amino acids with the exception of methionine, whereas xylanase/amylase did not exhibit significant differences in amino acid digestibility for any of the evaluated amino acids when compared to the control diets. The amino acids with the greatest digestibility response to xylanase/amylase/protease were cysteine (+5.4%), theonine (+4.4%), glycine (+3.6%), and valine (+3.3%), whereas the least responsive amino acids were methionine (+1.0%), glutamine (+2.0%), lysine (+2.0%), and arginine (+2.1%; Figure 1).
To further explore the reasons for the divergence in the digestibility response to xylanase/amylase/protease of different amino acids, the response relative to the undigested fractions of each amino acid in the control diets was analysed (Figure 2). Interestingly, the amount of individual undigested amino acids at the ileal level clearly determined the amino acid digestibility response to proteases on top of carbohydrases.
Figure 2 - Uplift of ileal amino acid digestibility of 21-d broiler chickens relative to the amounts of undigested amino acids at the ileal level, in response to carbohydrases or carbohydrases plus a protease in corn/soy or corn/soy/corn DDGS diets. Each point represents measured values for one amino acid in two trials.
Irrespective of the amino acid, a strong linear relationship between the amount of undigested amino acids and the digestibility response to enzymes was evident for xylanase/amylase/protease (R2=0.94 and 0.96). These results suggested that protein hydrolysis catalysed by the exogenous protease was responsible for the improvement of apparent ileal digestibility of amino acids. The effect of protease was non-specific to individual amino acids or diet types in this experiment, but it was mostly dependent on the inherent digestibility of amino acids in the diet.
Therefore, the contribution of protease to the digestibility amino acids that are very well digested will be smaller than that for amino acids that are less well digested. It becomes clear that providing greater amounts of highly digestible synthetic amino acids, such as DLmethionine to a diet, must reduce the potential increment in methionine digestibility from a protease. In contrast, amino acids with high concentration or low digestibility, such as glutamic acid, present higher increments on ileal amino acid digestibility from the protease. A conservative approach when recommending matrix values or down-specifications of limiting amino acids for protease supplementation appears to be preferable. Furthermore, digestibility improvement values produced in in vitro systems, or in vivo systems assessing the digestibility improvements of single ingredients, may overestimate the response of limiting amino acids if the absorption of these amino acids was not properly modelled.
III. ADDITIVE EFFECTS OF PROTEASE AND OTHER DIETARY ENZYMES
Effects of protease on poultry diets do not appear to be completely limited to protein digestion, but can also affect the digestibility of other nutrients. McAllister (1993), for example, reported increased digestion of corn starch with the use of a serine protease in a rumen in vitro model, which the author attributed to the disruption on the protein matrix in starch granules. Similarly, protein digestibility can also be affected by the presence of other dietary enzymes. Effects of carbohydrases and phytases on amino acid digestibility have been demonstrated and appear to involve a reduction of endogenous amino acid losses (Cowieson et al., 2008; Rutherfurd et al., 2007). It has also been suggested that phytases reduce the association of phytate and protein in the gizzard and proventriculus, increasing protein solubility (Yu et al., 2012). However, as effects of proteases and other dietary enzymes are all dependent on the amount of undigested amino acids present in the digestive tract, increments in amino acid digestibility from different enzymes cannot be additive. Therefore, the nutrient contribution from protease and other dietary enzymes in practical diets should not be determined in isolation.
Romero et al. (2012) conducted a series of studies to better understand the complex interactions of protease with different dietary ingredients and other enzymes. Two studies with 432 21-day or 288 42-day-old Ross-308 broiler males evaluated changes on the ileal energy contribution of substrates in response to xylanase and amylase without, or with protease in four broiler diets. The studies used a 2 x 2 x 3 factorial arrangement of treatments with two base grains (corn-soybean-meal; or wheat-soybean-meal diets); two fibrous protein ingredient levels (with, or without 10% corn-DDGS and 5% canola meal); and three enzyme levels. At 12 d or 32 d, three enzyme levels were applied: a negative control (NC); NC with xylanase from T. reesei and amylase from B. licheniformis; or NC with xylanase from T. reesei, amylase from B. licheniformis, and protease from B. subtilis (Axtra XAP; Danisco Animal Nutrition, DuPont Industrial Biosciences). At 21 d or 42 d, birds were euthanised; ileal digesta was collected, pooled per cage, and analysed to determine the apparent digestibility of energy, starch, fat, and protein. The increment of ileal energy digestibility of starch, fat, and protein was calculated as the mean change on the coefficient of apparent ileal digestibility of the enzyme treatment compared to the respective control treatment, and multiplied by the measured nutrient content in the diet and the assumed gross energy content of each substrate (starch=4.2 kcal/g; fat=9.4 kcal/g; protein =5.5 kcal/g).
Starch digestibility increased with xylanase/amylase (97.8% at 21 d; 96.6% at 42 d) and xylanase/amylase/protease (97.9% at 21 d; 97.0% at 42 d) compared to the NC (96.3% at 21 d; 93.4% at 42 d) across diets. There were no differences between xylanase/amylase and xylanase/amylase/protease on ileal starch digestion. Xylanase/amylase (84.4%) and xylanase/amylase/protease (85.8%) gradually increased protein digestibility (P < 0.05) at 21 d (NC=82.7%); but only xylanase/amylase/protease (85.1%) increased protein digestibility compared to the NC (82.4%) at 42 d. Both xylanase/amylase (83.3%) and xylanase/amylase/protease (84.0%) increased fat digestibility compared to the NC (80.2%) at 21 d. At 42 d, xylanase/amylase (86.6%) increased fat digestibility compared to NC (86.6%); and xylanase/amylase/protease (89.4%) further increased fat digestibility compared to xylanase/amylase.
Figure 3 - Improvement in ileal digestible energy due to supplemental enzymes (◊) and calculated ileal energy contribution from starch, fat and protein fractions (bars) in broiler chickens at 21 (A) and 42 (B) days of age. CS=corn/soybean meal-based diet; WS=wheat/soybean meal-based diet; HFI=high-fibre ingredients (corn-DDGS and canola meal); XA=xylanase and amylase; XAP=xylanase, amylase and protease enzymes.
At 21 d (Figure 3), the largest contributor to the increase in apparent ileal digestible energy (AIDE) with xylanase/amylase/protease was the protein fraction, and the protein contribution to digestible energy was greater in wheat than in corn-based diets. For xylanase/amylase, there was a similar trend with the exception of corn-based diets without high fibre ingredients such as corn-DDGs, where the protein contribution to the effect of the enzyme was relatively low. Evidently, fat digestibility had a more prominent role in the total energy effect in response to enzymes in wheat-based diets, whereas the contribution of fat digestibility in corn-based diets was marginal. The extent whereby increments in starch digestibility contributed to the energy contribution of xylanase/amylase or xylanase/amylase/protease was also different between diets, being greater in wheat- than in corn-based diets. At 42 d (Figure 3), the energy contribution from increments in protein digestibility was still relatively high compared to that of starch and fat. Energy contributions from fat were high in 21 d old chickens, but substantially less at 42 d. Only marginal improvements on the energy contribution from starch and fat due to protease were present at 42 days; however, they represented between 9 and 38 kcal/kg depending on the diet type.
Interestingly, the measured energy improvement from enzymes in corn-based diets closely resembled the sum of the contributions from digested starch, fat, and protein at 42 d. However, in wheat-based diets there was a consistent difference between the measured increase in ileal digestible energy from enzymes and the calculated contributions from starch, fat, and protein. This suggests that the digestibility of other components in the diet (other than fat, starch, and protein) may have contributed to the increased AIDE improvements due to enzymes. Enzyme inclusion may have caused either an increased fermentation of NSPs or the absorption of pentose sugars in the small intestine. The fact that this difference was not present in 21 d, but only in 42 d chickens also suggests interactions with the microbial populations of older birds may have occurred, with a resultant increase in digestion of fibre in the small intestine.
These data demonstrate that, although the main effect of protease was an improvement of protein digestibility, carbohydrases also significantly contributed to protein digestibility at least in some diets and for some growth periods. Similarly, proteases can also contribute to increasing the digestibility of other nutrients and presumably the digestion of NSPs. Other studies have also found overlapping effects of protease and carbohydrases on the digestibility of fat and starch in broiler chickens (Kalmendal and Tauson, 2012). Variability in the digestible nutrient response that is frequently seen with individual enzymes may be ameliorated by an integrated approach to exogenous enzyme utilisation which does not attribute independent additive nutrient contributions to single enzyme activities. It must also be stressed that the interactions of carbohydrases and proteases are likely to be dietdependent and carbohydrase enzyme effects may differ depending on the nature of the NSPs in different grains and vegetable protein ingredients.
IV. PROTEASE EFFECTS ON FIBRE DIGESTION IN CHICKENS
Effects of exogenous proteases on the digestion of fibre in in vitro rumen systems have been reported (Colombatto and Beauchemin, 2009). However, specific effects of protease on the digestion of fibre in poultry are not well understood. Olukosi and Romero (2012) recently studied the effects of different protease doses, with or without carbohydrases on the nutrient digestibility and the NSP flow of broiler chickens fed corn/soybean-meal diets with the inclusion of corn-DDGS. A total of 336 1-d old broilers received a standard broiler starter diet until day 14 when they were allocated to seven treatments in a randomised complete block design. Each treatment had 8 replicate cages with 6 birds per replicate cage. Diet 1, the control, contained no enzyme, diets 2 and 3 contained protease from B. subtilis at graded levels (protease 1; 5000 or 10000 u/kg), diet 4 contained another bacterial protease (protease 2; 10000 u/kg) whereas diets 5, 6 and 7 contained admixture of xylanase from T. reesei, amylase from B. licheniformis, and protease from B. subtilis (Axtra XAP; Danisco Animal Nutrition, DuPont Industrial Biosciences) at graded levels (50%, 100% and 200% the recommended dose for broilers, containing 2500, 5000, or 10000 protease u/kg). The diets were fed for 7 days, excreta samples were collected for the last 3 days of the experiment and ileal digesta samples were collected on the last day of the study.
The total tract flows of arabinose, xylose, galactose, glucose, and glucuronic acid from the NSP fraction (Figure 4) were reduced at the higher doses of protease, and both the intermediate and the high dose of xylanase/amylase/protease. In the specific case of xylose and arabinose, which are the main sugar components of arabinoxylans, the inclusion of xylanase and amylase generally reduced the flow of these sugars compared to protease alone at a comparable dose. These data suggest that fibre degradation may be one of the mechanisms by which proteases increase the digestion of nutrients in chickens. Although the reason of these effects of protease on NSP digestion is not known, studies in ruminant models have suggested that the use of proteases can disrupt cell wall associated proteins, which facilitates microbial colonisation of the substrate (Colombatto and Beauchemin, 2009). However, effects of exogenous proteases on the gut microbial populations of chickens have not been properly studied.
Figure 4 - Total tract flow of total sugars from the non-starch polysaccharides (NSP) fraction in 21-d-old broilers fed corn/soybean meal diets supplemented with different doses of two bacterial proteases or a combination xylanase, amylase, and protease (XAP).
V. OTHER FACTORS AFFECTING THE BIO-EFFICACY OF PROTEASES
Effects of feed proteases that are not directly related to nutrient digestibility have also been reported in the literature. For instance, Caine et al. (1998) reported a reduction in the level of trypsin inhibitors of soybean meal with the use of a serine protease, which may be present in practice and has not been fully recognised as one of the effects of value for poultry diets. Positive effects of exogenous proteases on the ability of birds to cope with intestinal disease challenges like Eimeria infections or necrotic enteritis have also been suggested, although mechanisms are not well understood and the evidence available is not definitive. Peek et al. (2009) found that a protease from Bacillus licheniformis increased the body weight gain of broilers challenged with three Eimeria species, and suggested that the mechanism was a reduction in the attachment of parasites to the mucus layer. This was supported by an increase in the thickness of the adherent mucus layer of the intestine due to protease. Yan et al. (2011) suggested that a protease avoided the growth of Clostridium perfringens in birds challenged with an Eimeria vaccine through improved absorption of protein and a reduction in the protein available for bacterial growth. However, other reports have not found clear effects of dietary protease on birds challenged with Eimeria vaccines (Walk et al., 2011). Nonetheless, intestinal health appears to be a factor that affects the animal performance responses in the field with the use of proteases, and require further study.
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