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What more do we need to know to optimize the use of a protease? Roselina Angel Department of Animal and Avian Sciences University of Maryland College Park, MD, USA Road map • Lessons from the field • May help us understand limits to protein digestion • Possible areas for enhancing protease efficacy • Why do we see the effects that we see? • • • • Stepwise digestion of protein Proteins resistant to digestion Importance of pH and passage rate Endogenous losses and sources of undigested protein • Nutrient-enzyme interactions • Impact of a protease on ingredient AA digestibility Impact of water pH and age on intestinal segment pH and nutrient digestibility Effect of water pH (5.8) on pH in intestinal segments Jejunum 8 d - 6.4 38 d - 6.4 Ileum 8 d - 6.6 38 d - 6.8 Ceca 8 d - 6.7 38 d - 6.1 Crop 8 d - 6.0 38 d - 6.1 Proventriculus 8 d - 1.9 38 d - 1.4 Gizzard 8 d - 2.9 38 d - 2.2 Duodenum 8 d - 5.8 38 d - 5.5 Angel et al., 2013- 6 replicates of 3 or 4 broilers/rep, at 8 and 38 d of age Effect of water pH (5.8 and 8.1) on pH in intestinal segments Jejunum 8 d 6.4/6.6 38 d 6.4/6.5 Ileum 8 d 6.6/7.1 38 d 6.8//7.0 Ceca 8 d 6.7/7.3 38 d 6.1/6.9 Crop 8 d - 6.0/7.6 38 d - 6.1/7.5 Proventriculus 8 d 1.9/4.1 38 d 1.4/2.9 Gizzard 8 d 2.9/5.6 38 d 2.2/4.6 Duodenum 8 d 5.8/6.0 38 d 5.5/5.7 Angel et al., 2013- 6 replicates of 3 or 4 broilers/rep, at 8 and 38 d of age Effect of water pH on nutrient digestibility Jejunum 8 d - 6.4/6.6 38 d - 6.4/6.5 Crop 8 d - 6.0/7.6 38 d - 6.1/7.5 Proventriculus 8 d - 1.9/4.1 38 d - 1.4/2.9 Gizzard 8 d - 2.9/5.6 38 d - 2.2/4.6 Duodenum 8 d - 5.8/6.0 38 d - 5.5/5.7 (Angel et al., 2013) Ileum 8 d - 6.6/7.1 38 d - 6.8//7.0 Ceca 8 d - 6.7/7.33 38 d - 6.1/6.9 Apparent ileal DM digestibility 84 85.1 a 5.8 pH 8.1 pH 82.3 82 a % 80 78.7 78 76 73.9 74 N =6 , 3 or 4 broilers/rep at 9 and 38 d of age, respectively c 72 SEM 2.3 8d Age 38 d b Effect of water pH on nutrient digestibility Ileum 8 d - 6.6/7.1 38 d - 6.8//7.0 Jejunum 8 d - 6.4/6.6 38 d - 6.4/6.5 Crop 8 d - 6.0/7.6 38 d - 6.1/7.5 (Angel et al., 2013) Ceca 8 d - 6.7/7.33 38 d - 6.1/6.9 Proventriculus 8 d - 1.9/4.1 38 d - 1.4/2.9 Gizzard 8 d - 2.9/5.6 38 d - 2.2/4.6 90 Duodenum 8 d - 5.8/6.0 38 d - 5.5/5.7 Apparent ileal protein digestibility 5.8 pH 8.1 pH 85.1 a 80 % 80.1 74.2 c 70 60 55.2 d N =6 , 3 or 4 broilers/rep at 9 and 38 d of age, respectively 50 SEM 2.1 8d Age 38 d b STEPS in protein digestion • Gastric area: Acid unfolding/Pepsin digestion – Polypeptides, oligopeptides • Intestinal luminal: pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidases) – Oligopeptides (2 to 6 aa), free amino acids • SI Mucosal cells – peptidases (membrane bound) – Di and tri peptides, free amino acids • Intraluminal – cytosolic peptidases – Free AA Role of gastric acidity • Denature (unfold) proteins to allow access for enzymes to hydrolyz • Extent of protein hydrolysis in gizzard correlated to time of exposure to pepsin/HCl (Keller, 1968) • Any inhibitions to this first step will partially inhibit subsequent steps – Type of protein – Acidity in gastric area Resistant protein in soybeans (Wang et al., 1995) • High molecular fraction (HMF) resulting from in vitro digestion of soybean protein with pepsin and pancreatin – when fed to rats • Resistant to further digestion – high homology between AA pattern of HMF and AA in feces • Bound to bile acids results in increased bile acid secretion • Increased cholesterol excretion • Increased N and fat excretion Proteins resistant to digestion (refractory) • Soybean proteins – Glycinin – accounts for 50+ % of SB proteins • Varies with cultivar – High in disulfide groups – 5 subunit polypeptides tightly bound by disulfide bridges Proteins resistant to digestion • Soybean proteins – Glycinin – 10.3% of glycinin (portions) fed to pre ruminant calves was still present in the distal ileum (Tukur et al., 1993) – From heat treated soy flour (for milk replacers) • Implications – In an 18% protein poultry diet containing 20% SBM (48% protein) – Would result in a 2.5 to 3% reduction in protein digestibility Proteins resistant to digestion • Sunflower – 2S albumin proteins – high in cystine • 13 subunits • SFA8 subunit highest in sulfur AA • Both SFA8 and LTP subunits bind lipids – Both subunits resist in vitro digestion (Berecz et al., 2013) • Association with lipids decreases lipid digestion • Quantification has not been done Proteins resistant to digestion • Wheat – Gliadin (and related proteins from rye and barley) very rich in proline • 13 subunits • SFA8 subunit highest in sulfur AA • Both SFA8 and LTP subunits bind lipids – Proline rich residue difficult to digest (Hausch et al., 2002) • Supplementation of a prolyl-edopepdidase improved digestibility of proline rich olygopeptides Effect of water pH intestinal pH Crop 8 d - 6.0/7.6 38 d - 6.1/7.5 (Angel et al., 2013) Ileum Impact of pH pepsin activity and stability 8 d - on 6.6/7.1 Jejunum 8 d - 6.4/6.6 38 d - 6.4/6.5 38 d - 6.8//7.0 (From Piper and Fentone, 1965) Ceca 8 d - 6.7/7.33 38 d - 6.1/6.9 Proventriculus 8 d - 1.9/4.1 38 d - 1.4/2.9 Gizzard 8 d - 2.9/5.6 38 d - 2.2/4.6 Duodenum 8 d - 5.8/6.0 38 d - 5.5/5.7 N =6 , 3 or 4 broilers/rep at 9 and 38 d of age, respectively Effect of age on pH of the proventriculus Bowen and Waldroup, 1969 Jimenez-Moreno et al., 2010 From Rynsberger, 2009, Mean of 10 b/age Impact of diet Ca and age on gizzard content pH (Corn-SBM St diet, no added inorganic P) pH at 5 d of age pH at 32 d of age 5.0 5.0 y = 1.6649x + 2.6552 R² = 0.7071 4.5 4.0 4.5 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 SEM 0.042 1.0 0 0.2 0.4 y = 1.1893x + 1.7993 R² = 0.447 4.0 0.6 tP = 0.42%, PP = 0.30%, nPP = 0.12% Limestone added Analyzed Ca 0.19 to 0.95 0.8 SEM 0.068 1.0 1 0 0.5 n=8 Angel et al., Unpublished 1 Impact of age on protein digestibility Protein digestibility, chickens 95 % 90 85 Batal and Parsons, 2002 Chickens, corn-SBM diet 80 75 4d 7d 10 d 14 d 21 d Noy and Sklan, 1997; Corn-SBM diet WHY? Sites of protein digestion and absorption Net Digestion and absorption Hurwitz et al., 1972 • Effect of dietary Ascaridia galli infection (most parasites in upper SI) • Effect of SI segment • SBM/Milo diet • 20.4 vs 14.4 % protein diets • White leghorn males– Infected at 1 week of age • Fed experimental diets for 5 days, starting at 5 weeks of age Hurwitz et al, 1972 Upper Jejunal digestion No infection @ 60% Infected @60% Duodenal digestion Non infected @ 8% Infected @50% Non infected Infected with Ascardia galli Upper Jejunal absorption No infection @ 40% Infected @40% Duodenal absorption Non infected @ -7% Infected @ -57% AA or peptide absorption • Different mechanisms of absorption • High competition for absorption of free AA – 5 transporter systems (Broer, 2008) – Transporter systems mostly Na dependent • More efficient and more rapid absorption of peptides Sources of protein entering the intestine Protein in the GIT – origin – feed or endogenous? How much and where digested? Pigs (from Fuller and Reeds, 1998) Feed N – Endogemous N – 35.3 grams/day 16.1 grams per day Stomach Endogenous N secretions g/d Feed N 5.3 N entering the GIT = 2.2 g feed N: 1 g endogenous N Jejunum 8.9 Ileum Large Intest. 1.9 Undigested 2.6 (7.4%) 35.3 1.6 (10%) Absorbed N Feed 28.2 (80%) 4.3 (12.6%) Endogenous 10.4 (65%) 4.1 (25%) Basal endogenous losses • Diet Independent endogenous flow decreases with age, chicks All AA -g/kg of DM intake Adedokun et al., 2007 Endogenous losses with or without a protease mg of AA/ kg DM Feed intake 800 Mateos et al., 2014 700 Mateos et al., 2014 + * * * 600 500 400 300 200 100 Thr Val Met Cys Lys Iso Gly Pro Rate of passage pH and mean retention time digesta in the GIT broilers 22 d of age (mean 25 broilers, min/max) (Angel et al., 2013) Mean feed particle size 0.822 mm (corn/SBM diet) Jejunum 6.1 (5.8/6.6) Ileum 6.5 (6.2/7.1) Ceca 6.4 (5.9/6.9) Crop 5.2 (4.3/5.9) Large intestine 6.4 (6.0/7.3) 5.0 (4.5-5.4) Proventriculus 1.6 (1.15/2.46) 6.3 (5.9-6.6) Gizzard 2.5 (1.6/3.2) Duodenum 6.0 (5.5/6.4) Total residence time as affected by age 10 d = 3h 15 min (2:32 -3:51 ) (SEM:09) 22 d = 4 h 25 min (3:10 – 4:42) (SEM:14) 30 d = 4 h 44 min (3:30 – 5:32) (SEM:19 42 d = 5 h 10 min (4:09-6:05) (SEM:25) pH and mean retention time digesta in the GIT broilers 22 d of age (mean 25 broilers, min/max) (Angel et al.,2013) Mean feed particle size 0.822 mm (corn/SBM diet) Jejunum 6.1 (5.8/6.6) D+J = 87 min Crop 5.2 (4.3/5.9) 12 min Ileum 6.5 (6.2/7.1) 80 min Ceca 6.4 (5.9/6.9) Large intestine 6.4 (6.0/7.3) Proventriculus 1.6 (1.15/2.46) 37 min ) Gizzard 2.5 (1.6/3.2) Total residence time as affected by age Duodenum 6.0 (5.5/6.4) 10 d 22 d 30 d 42 d = 3h 15 min (2:32 -3:51 ) (SEM:09) = 4 h 25 min (3:10 – 4:42) (SEM:14) = 4 h 44 min (3:30 – 5:32) (SEM:19 = 5 h 10 min (4:09-6:05) (SEM:25) Retrograde movement of digesta • Ocurrs in 3 areas (Duke, 1994) – a) Between the proventriculus and gizzard – b) From the ileum/jejunum through the duodenum the gizard/proventriculus – c) From the cloaca to ceca and proximal ileum Retrograde movement of digesta (Sklan et al., 1978) • Injected a radioactive isotope into bile ducts – Within 2 min – of injected dose – 40% in gizzard (50% still there after 20 min) – 30% in duodenum • Injected into upper Jejunum – 20% in gizzard within 2 min • Trypsin and lipase found in gizzard content – were still active when content incubated at 6.5 pH Relationship of passage rate and digestibility • Correlation between slower passage rates and increased nutrient digestibility poultry (Amerah et al., 2007; Svihus, 2011) • As passage rate slows, dry matter digestibility increase in gestating sows (Kim et al., 2007) • Fast emptying of the stomach resulted in decreased protein digestibility in calves (Toullec and Lalles, 1995; Lalles et al., 1999) Endogenous enzymes and nutrient interactions • Effect of feeding raw or heated SBM (Sklan et al., 1975) – Increases bile acid secretion 2X 90 • But partially reabsorbed in lower SI • 40% greater excretion in raw SBM 85 % fed chicks 80 – No effect on lipase activity at any 75 point in the SI 70 Free fatty acid net retention Heated SBM Raw SBM P<0.05 Endogenous enzymes and Nutrient interactions • Effect of feeding raw or heated SBM (Sklan et al., 1975) – Net absorption of fatty acids • 87.0 vs 75.6% • Complexation of FFA with undigested protein Triglycerides (polypeptides/oligopeptides) (Johnson, 1963; Sklan et al., 1975) Free fatty acids (FFA) • Importance of understanding nutrient interactions Diglycerides Monoglycerides Phospholipids Impact of ProAct on diet and ingredient digestibilities Reference method (apparent) Bertichini et al 2009 Full fat soy vs SBM % undigestible AA 45 % 40 35 30 25 20 15 10 5 0 -5 Full fat Soy SBM Improvement in essential apparent AA digestibility with 200 ppm protease 12 % improvement SBM Full fat soy 10 8 6 4 2 0 Met Lys Thr Cyst Ile Bertechini et al. (2009 ) Arg Val Phen Leu Soybean Meal Nutrient (analyzed) Bertichini et al., 2009 Angel, et al., 2010 Fat (%) 3.59 2.88 Moisture (%) 10.74 8.8 C. Protein (%) 44.44 48.0 Urease, pH change 0.03 0.01 Solubility KOH, % 88.15 89.6 (87 to 94.2) SBM- Amount of undigested AA (W/O ProAct) 45 40 % 35 30 25 20 15 10 5 0 Bertechini t al, 2009 Ref method (apparent) Angel et al., 2010 NDF (Standardized) SBM– percent of undigested – digested by ProAct 40 35 % 30 25 20 15 10 5 0 -5 Bertechini t al, 2009 Ref method (apparent) Angel et al 2010, NDF (True) SBM - Standardized ileal (SI) AA digestibility with 0 or 200 ppm of a commercial protease (Angel et al 2011) 92 Broiler Turkey Layer % digestibility 87 82 77 72 Met Lys Thr Cyst Ile Arg Val SBM – Improvement in SI AA digestibility with use of 200 ppm of a commercial protease (Angel et al 2011) % digestibility improvement 11 9 * * * * Broiler Turkey * Layer * 7 * 5 * * * * * 3 1 -1 -3 Met Lys Thr Cys Iso Arg Val Try How can we improve protein digestion/absorption? • What should be the target of an exogenous protease? • Unclear what the rate limiting step is in digestion • Ingredient/diet related • BUT there is information that allows us to potentiate the effect of proteases How can we improve protein digestion/absorption? • BUT there is information that allows us to potentiate the effect of proteases – Look at water pH/diet buffering capacity • Lowering pH, especially in young birds, can help with protease efficacy – Look at particle size, fiber concentration and type as means of decreasing gastric pH and reducing passage rate How can we improve protein digestion/absorption? • BUT there is information that allows us to potentiate the effect of proteases – Look closely at what ingredients are in the diet • Make sure NSP enzymes are present when needed – impacts on digestibility of all other nutrients • Use of phytases will increase the proteins available for digestion – interactions with proteases? • Make sure fat levels are not to low or to high – will change passage rate, and potentially interact with other digestive processes How can we improve protein digestion/absorption? • BUT there is information that allows us to potentiate the effect of proteases • Understand your ingredients – Low digestibility nutrients – higher potential for enzyme to work – Apply meta analysis knowledge – What diets (ingredients) and with what characteristics will have the greatest potential for a protease to work on – Diet Ca concentrations and solubility – Refractory proteins in ingredients are potential feed proteins that can be digested by exogenous proteases How can we improve protein digestion/absorption? • BUT there is information that allows us to potentiate the effect of proteases • Understand the impact of diet (ingredient) on endogenous losses – Anti nutrient content (trypsin inhibitors, phytate, …), fiber, … • Pay attention to AA balance – essential vs. non essential amino acids • Look for balance in aa and peptide residue for absorption (not looking for all AA as a result of protein digestion) How can we improve protein digestion/absorption? • BUT there is information that allows us to potentiate the effect of proteases • Feed back mechanisms of nutrients on enzyme secretion • How does it change endogenous losses • Costs of digestion • What is really the N portion that is undigested • Endogenous • Decrease endogenous losses (fiber, anti nutrients, exogenous enzymes, ..) • Feed proteins – What feed proteins are poorly digested? Remember factors affecting digestion • Microbial populations • Host-diet-microbial population interactions • Effect overall and intestinal health • Affect how diets are digested, nutrients absorbed and utilized • More than enzymatic • • • • Accessibility of substrate to enzymes (“caging”) Interactions between nutrients (complexations) Interaction between exogenous enzymes Interactions between diet/endogenous enzymes/exogenous enzymes Thank you! Questions?