What more do we need to know to optimize the use of a protease?

Transcription

1 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

2 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

3 Impact of water ph and age on intestinal segment ph and nutrient digestibility

4 Effect of water ph (5.8) on ph in intestinal segments Jejunum 8 d d Ileum 8 d d Crop 8 d d Ceca 8 d d Proventriculus 8 d d Gizzard 8 d d Duodenum 8 d d Angel et al., replicates of 3 or 4 broilers/rep, at 8 and 38 d of age

5 Effect of water ph (5.8 and 8.1) on ph in intestinal segments Jejunum 8 d 6.4/ d 6.4/6.5 Ileum 8 d 6.6/ d 6.8//7.0 Crop 8 d - 6.0/ d - 6.1/7.5 Ceca 8 d 6.7/ d 6.1/6.9 Proventriculus 8 d 1.9/ d 1.4/2.9 Gizzard 8 d 2.9/ d 2.2/4.6 Duodenum 8 d 5.8/ d 5.5/5.7 Angel et al., replicates of 3 or 4 broilers/rep, at 8 and 38 d of age

6 Effect of water ph on nutrient digestibility (Angel et al., 2013) Crop 8 d - 6.0/ d - 6.1/7.5 Proventriculus 8 d - 1.9/ d - 1.4/2.9 Gizzard 8 d - 2.9/ d - 2.2/4.6 Duodenum 8 d - 5.8/ d - 5.5/5.7 Jejunum 8 d - 6.4/ d - 6.4/ % Ileum 8 d - 6.6/ d - 6.8//7.0 Ceca 8 d - 6.7/ d - 6.1/ a Apparent ileal DM digestibility 5.8 ph 8.1 ph 82.3 a 78.7 b c N =6, 3 or 4 broilers/rep at 9 and 38 d of age, respectively 72 SEM d Age 38 d

7 Crop 8 d - 6.0/ d - 6.1/7.5 Effect of water ph on nutrient digestibility (Angel et al., 2013) Jejunum 8 d - 6.4/ d - 6.4/6.5 Proventriculus 8 d - 1.9/ d - 1.4/2.9 Gizzard 8 d - 2.9/ d - 2.2/4.6 Duodenum 8 d - 5.8/ d - 5.5/ Ileum 8 d - 6.6/ d - 6.8//7.0 Ceca 8 d - 6.7/ d - 6.1/6.9 Apparent ileal protein digestibility 5.8 ph 8.1 ph 85.1 a 80.1 b % c d N =6, 3 or 4 broilers/rep at 9 and 38 d of age, respectively 50 SEM d Age 38 d

8 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

9 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

10 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

11 Proteins resistant to digestion Soybean proteins (refractory) Glycinin accounts for 50+ % of SB proteins Varies with cultivar High in disulfide groups 5 subunit polypeptides tightly bound by disulfide bridges

12 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

13 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

14 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

15 Crop 8 d - 6.0/ d - 6.1/7.5 Effect of water ph intestinal ph (Angel et al., 2013) Jejunum 8 d - 6.4/ d - 6.4/6.5 Proventriculus 8 d - 1.9/ d - 1.4/2.9 Gizzard 8 d - 2.9/ d - 2.2/4.6 Duodenum 8 d - 5.8/ d - 5.5/5.7 Impact of ph on pepsin activity and stability Ileum 8 d - 6.6/ d - 6.8//7.0 (From Piper and Fentone, 1965) Ceca 8 d - 6.7/ d - 6.1/6.9 N =6, 3 or 4 broilers/rep at 9 and 38 d of age, respectively

16 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

17 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 y = x R² = y = x R² = SEM SEM tp = 0.42%, PP = 0.30%, npp = 0.12% Limestone added Analyzed Ca 0.19 to 0.95 n = 8 Angel et al., Unpublished

18 Impact of age on protein digestibility Protein digestibility, chickens 95 % Batal and Parsons, 2002 Chickens, corn-sbm diet 75 4 d 7 d 10 d 14 d 21 d Noy and Sklan, 1997; Corn-SBM diet WHY?

19 Sites of protein digestion and absorption

20 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

21 Hurwitz et al, 1972 Non infected Infected with Ascardia galli Upper Jejunal digestion No 60% Upper Jejunal absorption No 40% Duodenal digestion Non 8% Duodenal absorption Non -7% -57%

22 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

23 Sources of protein entering the intestine

24 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 N entering the GIT = 2.2 g feed N: 1 g endogenous N Stomach Jejunum Ileum Large Intest. Endogenous N secretions g/d Feed N 35.3 Undigested 2.6 (7.4%) 1.6 (10%) Feed Absorbed N Endogenous 28.2 (80%) 10.4 (65%) 4.3 (12.6%) 4.1 (25%)

25 Basal endogenous losses Diet Independent endogenous flow - decreases with age, chicks Adedokun et al., 2007 All AA -g/kg of DM intake

26 mg of AA/ kg DM Feed intake 800 Endogenous losses with or without a protease Mateos et al., 2014 Mateos et al., * * * Thr Val Met Cys Lys Iso Gly Pro

27 Rate of passage

28 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 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) Proventriculus 1.6 (1.15/2.46) 5.0 ( ) 6.3 ( ) Large intestine 6.4 (6.0/7.3) 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)

29 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 mm (corn/sbm diet) Jejunum 6.1 (5.8/6.6) D+J = 87 min Ileum 6.5 (6.2/7.1) 80 min Ceca 6.4 (5.9/6.9) Crop 5.2 (4.3/5.9) 12 min 37 min Proventriculus 1.6 (1.15/2.46) Gizzard 2.5 (1.6/3.2) Duodenum 6.0 (5.5/6.4) ) Large intestine 6.4 (6.0/7.3) 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)

30 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

31 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

32 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)

33 Endogenous enzymes and nutrient interactions Effect of feeding raw or heated SBM (Sklan et al., 1975) Increases bile acid secretion 2X But partially reabsorbed in lower SI 40% greater excretion in raw SBM fed chicks No effect on lipase activity at any point in the SI % Free fatty acid net retention Heated SBM Raw SBM P<0.05

34 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

35 Impact of ProAct on diet and ingredient digestibilities

36 Full fat soy vs SBM % undigestible AA Reference method (apparent) Bertichini et al % 40 Full fat Soy SBM

37 Improvement in essential apparent AA digestibility with 200 ppm protease 12 % improvement 10 SBM Full fat soy Met Lys Thr Cyst Ile Arg Val Phen Leu Bertechini et al. (2009 )

38 Soybean Meal Nutrient (analyzed) Bertichini et al., 2009 Angel, et al., 2010 Fat (%) Moisture (%) C. Protein (%) Urease, ph change Solubility KOH, % (87 to 94.2)

39 SBM- Amount of undigested AA (W/O ProAct) % Bertechini t al, 2009 Ref method (apparent) Angel et al., 2010 NDF (Standardized)

40 SBM percent of undigested digested by ProAct % Bertechini t al, 2009 Ref method (apparent) Angel et al 2010, NDF (True)

41 SBM - Standardized ileal (SI) AA digestibility with 0 or 200 ppm of a commercial protease (Angel et al 2011) 92 Broiler Turkey Layer % digestibility Met Lys Thr Cyst Ile Arg Val

42 SBM Improvement in SI AA digestibility with use of 200 ppm of a commercial protease (Angel et al 2011) % digestibility improvement * * * * * * * * * * * * Broiler Turkey Layer -3 Met Lys Thr Cys Iso Arg Val Try

43 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

44 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

45 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

46 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

47 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)

48 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?

49 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

50 Thank you! Questions?