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Advances in understanding_enzyme_substrates_in_feed_and_available_solutions_romero_afma_2015
- 1. Advances in Understanding Enzyme Substrates in Feed and Available Solutions Luis Romero, PhD October 14th, 2015
- 2. Outline Introduction. Mode of action and value of enzymes Substrate and enzymes interactions in the gut Phytase Carbohydrases Proteases The microbiota as a key player Capturing increased animal performance
- 3. Reduced performance + mortality There is a gap between genetic potential and commercial performance in animal production O Production frontier Meat(kg/flock) Feed, labor, other inputs ($) Sub-optimal production Sub-optimal diet digestibility Feed ingredient variation Sub-clinical disease Clinical disease Environmental stress Commercial performance Value Potential Romero, 2010
- 4. Mode of action of exogenous enzymes in animals is not limited to hydrolysis of one substrate 10/29/2015 Enzyme + substrate • Digesta • Physical structure • Solubility of nutrients • Host • Feedback mechanisms • Endogenous enzymes • Gut development • Immune response • Microbiome profile and function Balance and retention of nutrients
- 5. Screening Small scale testing Commercial application Customer need Assay and product specifications Understanding exogenous enzymes for animal nutrition Customer value Disrupting technologies Mode of action Systems biology
- 6. Substrate and enzymes interactions in the gut 10/29/2015
- 7. Characterization of phytase activity in-vitro Aspergillus niger E. coli Citrobacter Braakii Buttiauxella sp. Menezes-Blackburn et al. 2015. J. Agr. Food Chem.
- 8. Maximum IP degradation in the digestive tract is now reaching levels that are close to 90%, but multiple factors affect maximum disappearance 10/29/2015 * 0, 500 or 1000 FTU/kg of Buttiauxella sp. phytase were supplied to broilers from 11 to 13 d of age Li, Angel, et al. Submitted for publication, 2015 Factor Ca nPP PP Phytase Ca×nPP Ca×PP Ca× Phytase nPP×PP nPP× Phytase PP× Phytase Ca×nPP× PP Ca×nPP× Phytase Ca×PP× Phytase nPP×PP× Phytase Ca×nPP× PP×Phytas e F Prob 0.0003 0.86 <0.0001 <0.0001 0.66 0.22 0.38 0.17 <0.0001 0.0024 0.12 0.27 0.88 0.15 0.79
- 9. 1420 970 295 44 96 15 28 107 213 65 176 96 138 28 0 200 400 600 800 1000 1200 1400 1600 1800 Starch Protein Fat Arabinoxylans Pectins Cellulose Resistant oligo- saccharides Digestibleenergyinacorn-soy-DDGSdietinbroilerchickens andyoungpigs(kcal/kg) Digestible energy Non digestible energy Undigested energy and protein substrates 10/29/2015 Non digestible energy ~ 820 kcal/kg Realistic improvement ~ 440 Current improvement ~ 100 kcal/kg Non digestible protein ~ 40 g/kg Realistic improvement ~ 20 g/kg Current improvement ~ 4 g/kg
- 10. Mechanisms of action of carbohydrases and proteases in broiler diets Xylanase, beta-glucanase Reduced viscosity (Choct, 1999) Improved access to cell contents (Cowieson, 2005) Prebiotic effects (Fernandez et al., 2000) Possible reduction of endogenous inputs (Satchithanandam et al., 1990) Feed intake Digestion Feces Endogenous inputs Fermentation Absorption Production a.a., starch, fat a.a., NE A X X A P Protease Hydrolysis of dietary protein and increased protein solubility (Caine et al., 1998) Disruption of protein-starch interactions in corn (Mc Allister et al., 1993; Belles et al., 2000 ) Disruption of protein-fiber interactions (Pedersen, 2015) Amylase Down regulation of pancreatic amylase (Jiang et al., 2008) Augmentation of pancreatic amylase activity in young animals (Gracia et al., 2003) Improvement of digestion of resistant starch in corn and corn by products (Sharma et al., 2010) P X SCFA
- 11. Xylanases and beta-glucanases. Shifting the target from the soluble to the insoluble fractions Arabinoxylan and beta-glucan in some feed ingredients (% dry matter) Source: Choct (2006); Danisco Non Starch Polysaccharide (NSP) database (2012) 2 7 12 17 22 27 Corn Wheat Barley Rye Wheat bran Wheat DDGS Corn DDGS Soybean meal Rapeseed meal Sunflower cake Insoluble arabinoxylan Soluble arabinoxylan Total beta-glucan
- 12. It is not just about solubilisation of fibre; it is about net energy for the animal 10/29/2015 Enzymes: Solubilize fiber and release nutrients Amino acids & peptides Starch Fats More nutrients absorbed Microbial fermentation Availability of energy from fiber via SCFA production: • Butyrate (2185 kj/mol) • Propionate(1528 kj/mol) • Acetate(874kj/mol) Hemi- cellulose
- 13. There is considerable potential to increase protein digestibility in commercial diets 10/29/2015 Ingredient * Crude protein ME (Kcal/kg) Main proteins Key amino acids % App. dig** Poultry Swine Corn 8 0.82 3390 3350 Zein Leu, asp, glu Wheat 11 0.81 3210 3415 Glutenin Glu Soybean meal 48 0.85 2458 3140 Glycinin, beta- conglycinin Phe, tyr, leu Corn DDGS 27 0.65- 0.85 2800 3300 Globulin, glutelin, zein Leu, asp, glu Sorghum 11 0.68 3310 3230 Kafirin Pro, glu Canola meal 38 0.77 2110 2600 Cruciferin, napin Glu, asp Sunflower meal 41 0.83 2310 2740 Helianthinin Glu, asp Feather meal 85 0.50- 0.75 2880 2270 Keratin Ser, pro, gly Meat and bone meal 50 0.65- 0.80 2530 2435 Collagen Gly, ala, pro
- 14. Exogenous proteases Serine proteases (3.4.21.xx) Trypsin subfamily proteases (3.4.21.4) Subtilisin proteases (3.4.21.62) • Catalytic mechanism = His-Ser-Asp • Alkaline pH optimum • Higher specificity to negatively charged substrates • Higher specificity to hydrophobic substrates Aspartic acid proteases Metallo-protease Cysteine proteases Most commercially available proteases fall into this category
- 15. Proteomics - a new tool Proteomics is the large-scale experimental analysis of proteins present in a biological sample. It usually relies on the extraction of the proteins from the sample, their separation, protein digestion, followed by mass spectrometry analysis Proteomics has been applied to the study of food or feed protein digestion, and the terminology “protein digestomics” has been put forward by Picariello et al, 2013 • Le Gall et al. (2005) • Identified two proteins in peas, lectin and albumin PA1b, that were totally resistant to gastric and small intestinal digestion in pigs • Fisher et al. (2007) • Identified aggregated peptides of partly degraded β-conglycinin alpha subunits in the undigested ileal residue of pigs fed soybean meal 10/29/2015
- 16. Improvements on amino acid digestibility due to enzymes are proportional to ileal undigested amino acids in control diets 10/29/2015 y = 0.03x R2 = 0.56 y = 0.03x R2 = 0.42 y = 0.12x R2 = 0.96 y = 0.13x R2 = 0.94 -0.20 0.00 0.20 0.40 0.60 0.80 1.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Apparently undigested fraction of amino acid (g/kg) Apparentupliftonaminoacid digestibility(g/kg) XA-Corn/SBM XAP-Corn/SBM XA-Corn/SBM/DDGS XAP-Corn/SBM/DDGS metmet lys lys gln gln thr thr leu leu asn asn cys cys val val Romero et al., 2013
- 17. Protein contributed a significant amount of energy in response to enzymes, particularly protease, in 21-d broilers IDE = ileal digestible energy CS=Corn/Soy; WS=Wheat/Soy XA = xylanase and amylase; XAP = XA plus protease Mixed diets contained 10% corn DDGS and 5% canola meal 15 13 15 23 28 35 35 302 10 0 12 28 25 40 38 5 29 25 39 36 56 34 54 0 89 54 75 59 133 94 120 0 20 40 60 80 100 120 140 CS + XA CS + XAP CS Mixed + XA CS Mixed + XAP WS + XA WS + XAP WS Mixed + XA WS Mixed + XAP Improvementofilealdigestible energy21d(kcal/kgDM) Starch Fat Protein IDE Romero et al., 2015
- 18. At 42 days, starch contribution was relatively greater, and carbohydrase had smaller effects on protein contributions 23 30 38 34 38 50 67 78 4 20 8 18 11 20 24 34 25 30 21 40 18 42 0 20 40 60 80 100 120 140 160 180 CS + XA CS + XAP CS Mixed + XA CS Mixed + XAP WS + XA WS + XAP WS Mixed + XA WS Mixed + XAP Improvementofilealdigestible energy(kcal/kgDM) Starch Fat Protein Romero et al., 2015 IDE = ileal digestible energy CS=Corn/Soy; WS=Wheat/Soy XA = xylanase and amylase; XAP = XA plus protease Mixed diets contained 10% corn DDGS and 5% canola meal
- 19. Contributions of starch, fat and protein did not explain ileal digestible energy in wheat based diets at day 42 d 23 30 38 34 38 50 67 78 4 20 8 18 11 20 24 34 0 25 1 30 21 40 18 42 35 90 32 96 134 147 149 275 0 50 100 150 200 250 300 CS + XA CS + XAP CS Mixed + XA CS Mixed + XAP WS + XA WS + XAP WS Mixed + XA WS Mixed + XAP Improvementofilealdigestible energy(kcal/kgDM) Starch Fat Protein IDE Romero et al., 2015 IDE = ileal digestible energy CS=Corn/Soy; WS=Wheat/Soy XA = xylanase and amylase; XAP = XA plus protease Mixed diets contained 10% corn DDGS and 5% canola meal
- 20. Proteases can have significant effects on total tract NSP disappearance in broiler chickens * A subtilisin protease at two doses (P5000 and P10000) and a combination of xylanase, amylase and protease at three doses (XAP2500, XAP5000, XAP10000) were supplemented in a corn-based diet from 14 to 21 d to broiler chickens Olukosi et al, 2015
- 21. Some practical implications Net effects of enzymes on performance are greater in diets with greater undigested substrates, which normally correspond to diets with more fibrous ingredients. Effects of exogenous enzymes on the digestibility of different energy substrates overlap. Matrices are not additive. Therefore, it is always better to work with enzymes combinations with enough knowledge of mode of action and robust matrices. 10/29/2015
- 22. The microbiota as a key player 10/29/2015
- 23. 24 You are bacterial and so are your birds and pigs!
- 24. Avian gut microbiota Total bacterial pop. ~1014 cells (10x more than host cells”) The avian gastrointestinal tract consists of many small ecological niches; either mucosal or luminal Gut bacteria are in the mucus layer at the epithelium and on digesta forming biofilms Essentially sterile at hatch, the gut flora of an adult bird is relatively stable and difficult to change by feed additives Microbiota influences: • Development and function of immune system • Metabolism, appetite • Disease • Behavior
- 25. Critical interactions of exogenous enzymes and the gut microbiome Direct effect Critical interaction Feed enzymes Increased growth and feed efficiency More nutrients digested More nutrients absorbed and metabolizedReduced endogenous inputs for digestion Reduced populations of pathogenic bacteria Prebiotic effects Reduced endogenous inputs for immunity Less nutrients available for pathogens Improved gut integrity Increased fibre digestion
- 26. Enzymes might affect gut health through changes in the available substrate and direct effect in the mucosa • Xylanases have been shown to have pre-biotic effects in poultry (Fernandez, 2000) and other species through selective stimulation of beneficial bacteria and production of short-chain fatty acids (SCFA) (Broekaert et al., 2011) • Increased undigested protein appears to be a predisposing factor for dysbacteriosis related to necrotic enteritis (Dahiya et al., 2007) • Protease has been shown to improve performance of chickens challenged with Eimeria spp. (Peek et al, 2009)
- 27. Enteric disease is a limiting factor to the efficacy of exogenous enzymes due to mal-absoption 281 311 308 304 315 38 57 94 122 130 305 191 110 49 -0.9 0 100 200 300 400 500 600 700 800 0 0.5 1 1.5 2 Energyallocation (kcal/bird/day) Lesion scores (0-4) Maintenance cost Added energy lost in excreta Retained energy MEn intake Teeter et al. 2011; Broussard et al., 2008 Energy partitioning of 42-48 d old broilers challenged with oocysts of three Eimeria species
- 28. Combinations of enzymes and DFMs increase the consistency of response in diverse levels of challenge 10/29/2015 Dersjant-Li et al., 2014
- 29. The development of deep sequencing techniques offers completely new insights into the role of the gut microbiome 10/29/2015 Fraher et al., 2012 C Huttenhower et al. Nature 486, 207-214 (2012) doi:10.1038/nature11234 Carriage of microbial taxa varies while metabolic pathways remain stable within a healthy population
- 31. Capturing value in commercial conditions • Selection of enzymes should be based on the effects on undigestible substrates of base diets • Enzymes combinations with significant, measurable and reliable activity levels are preferable: • Wide and consistent range of functionalities • Higher chance of reliable net benefits in variable commercial conditions • Proactive management of nutrient interactions are necessary: • Effect of Ca on phytase activity • Estimation of AME and protein quality • Respond to seasonal or supply driven changes in ingredient quality • Optimization of gut health in critical 10/29/2015
- 32. Exogenous enzymes - Future R&D directions Increase research on application knowledge • Ingredient, animal and additive interactions • Improvements on in-vitro simulations Application of omics tools • Proteomics. Increased digestion of undigested proteins • Metagenomics. Increased energy from fibre; optimization of gut health • Metabolomics and transcriptomics. Intestinal and systemic mechanisms New applications of enzymes beyond digestion of nutrients 10/29/2015
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