Protein Digestion

1. Protein Digestion

2. Monogastric Protein Digestion • Whole proteins are not absorbed • Too large to pass through cell membranes intact • Digestive enzymes • Hydrolyze peptide bonds • Secreted as inactive pre-enzymes • Prevents self-digestion

3. Aromatic amino acids Monogastric Protein Digestion • Initiated in stomach • HCl from parietal cells • Stomach pH 1.6 to 3.2 • Denatures 40, 30, and 20 structures • Pepsinogen from chief cells • Cleaves at phenylalanine, tyrosine, tryptophan • Protein leaves stomach as mix of insoluble protein, soluble protein, peptides and amino acids HCl Pepsinogen Pepsin

4. Protein Digestion – Small Intestine • Pancreatic enzymes secreted • Trypsinogen • Chymotrypsinogen • Procarboxypeptidase • Proelastase • Collagenase Zymogens

5. Monogastric Digestion – Small Intestine • Zymogens must be converted to active form • Trypsinogen Trypsin • Endopeptidase • Cleaves on carbonyl side of Lys & Arg • Chymotrypsinogen Chymotrypsin • Endopeptidase • Cleaves carboxy terminal Phe, Tyr and Trp • Procarboxypeptidase Carboxypeptidase • Exopeptidase • Removes carboxy terminal residues Enteropeptidase/Trypsin Trypsin Trypsin

6. Protein Digestion • Small intestine (brush border) • Aminopeptidases • Cleave at N-terminal AA • Dipeptidases • Cleave dipeptides • Enterokinase (or enteropeptidase) • Trypsinogen trypsin • Trypsin then activates all the other enzymes

7. Trypsin Inhibitors • Small proteins or peptides • Present in plants, organs, and fluids • Soybeans, peas, beans, wheat • Pancreas, colostrum • Block digestion of specific proteins • Inactivated by heat

8. Protein Digestion • Proteins are broken down to • Tripeptides • Dipeptides • Free amino acids

9. Free amino acids Carrier systems Neutral AA Basic AA Acidic AA Imino acids Entrance of some AA is via active transport Requires energy Na+ Na+ Free Amino Acid Absorption

10. Amino Acid Transporters – Brush Border Membrane

11. Peptide Absorption • Form in which the majority of protein is absorbed • More rapid than absorption of free amino acids • Active transport • Energy required • Metabolized into free amino acids in enterocyte • Only free amino acids absorbed into blood

12. Absorption of Intact Proteins • Newborns • First 24 hours after birth • Immunoglobulins • Passive immunity • Adults • Paracellular routes • Tight junctions between cells • Intracellular routes • Endocytosis • Pinocytosis • Of little nutritional significance... • Affects health (allergies and passive immunity)

13. % Stoll et al. (1998) In the Enterocytes… • First cells that can use the amino acids • Transport into portal blood • Protein synthesis • Digestive enzymes • Structure and growth • Energy

14. Basolateral Membrane • Transport of free amino acids only* • Peptides are hydrolyzed within the enterocyte • Transport mainly by diffusion and Na-independent carriers Groff & Gropper, 2000 *Whole proteins are nutritionally insignificant...

15. Protein Transport in the Blood • Amino acids diffuse across the basolateral membrane • Enterocytes  portal blood  liver  tissues • Transported mostly as free amino acids • Liver • Breakdown of amino acids • Synthesis of non-essential amino acids

16. Overview of Protein Digestion and Absorption in Monogastrics Groff & Gropper, 2000

17. Ruminant Protein Digestion • Ruminants can exist with limited dietary protein sources due to microbial protein synthesis • Essential amino acids synthesized • Microbial protein is not sufficient during: • Rapid growth • High production

18. Protein in the Ruminant Diet • Types of protein: • Dietary protein – contains amino acids • Rumen Degradable Protein (RDP) – available for use by rumen microbes • Rumen Undegradable Protein (RUP) – escapes rumen fermentation; enters small intestine unaltered • Varies with diet, feed processing • Dietary non-protein nitrogen (NPN) – not true protein; provides a source of nitrogen for microbial protein synthesis • Relatively CHEAP - decreases cost of protein supplementation

19. Ruminant Protein Feeding • Feed the rumen microbes first (RDP) • Two counteractive processes in rumen • Degradation of (dietary) protein • Synthesis of microbial protein • Feed proteins that will escape fermentation to meet remainder of animal’s protein requirements • Escape protein, bypass protein, or rumen undegradable protein (RUP) • Aldehydes increase inter-protein cross-linking • Heat treatment • Utilization depends on • Digestibility of RUP source in the small intestine • Protein quality

20. Protein Degradation in Rumen

21. Rumen Protein Utilization • Factors affecting ruminal degradation • Rate of passage • Rate of passage   degradation  • Solubility in water • Must be solubilized prior to degradation • Heat treatment • Degradation  • N (and S) availability • Energy availability (carbohydrates)

22. Protein Fractions • Dietary proteins classified based on solubility in the rumen • A • NPN, instantly solubilized/degraded • B1 B2 B3 • Potentially degradable • C • Insoluble, recovered in ADF, undegradable

23. Ruminant Protein Digestion • Rumen microbes use dietary protein • Creates difference between protein quality in feed and protein actually absorbed by host • Microbes break down dietary protein to • Amino acids • NH3, VFAs, and CO2 • Microbes re-synthesize amino acids • Including all the essential amino acids from NH3 and carbon skeletons No absorption of protein or amino acids from rumen (or from cecum or large intestine!)

24. Protein Hydrolysis by Rumen Microbes • Process with multiple steps • Insoluble protein is solubilized when possible • Peptide bonds of solubilized protein are cleaved • Microbial endo- and exo-peptidases • Amino acids and peptides released • Peptides and amino acids absorbed rapidly by bacteria • Bacteria degrade into ammonia N (NH3) • NH3 used to produce microbial crude protein (MCP)

25. Microbial Crude Protein (MCP) • Protein produced by microbial synthesis in the rumen • Primary source of protein to the ruminant animal • Microbes combine ammonia nitrogen and carbohydrate carbon skeleton to make microbial crude protein • Diet affects the amount of nitrogen entering the small intestine as microbial crude protein

26. Factors Limiting Microbial Protein Synthesis • Amount of energy • ATP • Available nitrogen • NPN • Degraded feed intake protein nitrogen (RDP) • Available carbohydrates • Carbon residues for backbone of new amino acid Microbial crude protein synthesis relies on synchronization of carbohydrate (for carbon backbones) and nitrogen availability (for amino group)

27. VFA (CHO fermentation) Concentration Blood NH3 Rumen NH3 Time post-feeding Adapted from Van Soest, 1994 Microbial Protein Synthesis • Synchronization of carbohydrate and N availability • NPN supplementation • Carbohydrates used for carbon skeleton of amino acids Carbon backbone (from CHO fermentation)

28. Microbial Protein Formation Dietary Starch Sugar Dietary Cellulose Hemicellulose rapid slow Dietary NPN Carbon Skeletons Sulfur Other Co-factors rapid Microbial Proteins NH3 ATP Amino Acids slower very slow Dietary Insoluble RDP Dietary Soluble RDP

29. Nitrogen Recycling • Excess NH3 is absorbed through the rumen wall to the blood • Quickly converted to urea in the liver • Excess NH3 may elevate blood pH • Ammonia toxicity • Costs energy • Urea (two ammonia molecules linked together) • Relatively non-toxic • Excreted in urine • Returned to rumen via saliva (rumination important) • Efficiency of nitrogen recycling decreases with increasing nitrogen intake

30. Nitrogen Recycling • Nitrogen is continually recycled to rumen for reutilization • Ability to survive on low nitrogen diets • Up to 90% of plasma urea CAN be recycled to rumen on low protein diet • Over 75% of plasma urea will be excreted on high protein diet • Plasma urea enters rumen • Saliva • Diffuses through rumen wall from blood Urease Urea Ammonia + CO2

31. Feed Protein, NPN and CHO RUP Feed Protein AA Feed Protein RDP NH3/NH4 NH3 SMALL INTESTINE Feed NPN Bacterial N MCP MCP AA NH4+ loss RUMEN Salivary N Liver ATP Blood Urea

32. Ruminant Digestion and Absorption • Post-ruminal digestion and absorption closely resembles the processes of monogastric animals • However, amino acid profile entering small intestine different from dietary profile

33. Overview of Protein Feeding Issues in Ruminants • Rumen degradable protein (RDP) • Low protein quality in feed  very good quality microbial proteins • Great protein quality in feed  very good quality microbial proteins • Feed the cheapest RDP source that is practical regardless of quality • Rumen undegradable protein (RUP) • Not modified in rumen, so should be higher quality protein as fed to animal • May cost more initially, but may be worth cost if performance boosted enough

34. Recycled urea Salivary Urea NH3 UREA LIVER NPN Non-utilized Ammonia Dietary Nitrogen PEPTIDES NH3 AMINO ACIDS LEVEL TO PROVIDE FOR MAXIMUM MICROBIAL GROWTH POOL AMINO ACIDS 65% OF PROTEIN AMINO ACIDS RDP PROTEIN MICROBIAL PROTEIN SMALL INTESTINE 35% OF PROTEIN RUP Reticulo-rumen

35. Functional Feeds Functional feeds may be defined as any feed or feed ingredient that produces a biological effect or health benefit that is above and beyond the nutritive value of that feedstuff Many feeds and their components fit this definition

36. Functional Proteins Functional proteins are feed-derived proteins that, in addition to their nutritional value, produce a biological effect in the body

37. Feedstuffs with Biologically Active Proteins Milk Colostrum Whey Protein Concentrates/Isolates Plasma or serum Other animal-derived feedstuffs Fish meal Meat and bone meal Fermented animal-based products Yeast Lactobacillus organisms Soy products

38. Protein Size Affects Function Many protein hormones are functional even when fed to animals thyrotropin-releasing hormone (TRH, a 3-amino acid peptide) luteinizing hormone-releasing hormone (LHRH, a 10-amino acid peptide) insulin (a 51-amino acid polypeptide) The smaller the peptide, the more “functional” it is when fed 100% activity for TRH, 50% for LHRH, and 30% for insulin Feedstuffs containing protein hormones (colostrum) have biological activity when fed to animals

39. Production of Bioactive Peptides From Biologically-Inactive Proteins Peptides produced from intact inactive proteins by incomplete digestion via proteases in stomach and duodenum or via microbial proteases in rumen Many of these biologically active peptides (typically 2-4 amino acid residues) are stable from further digestion Some peptides bind to specific epithelial receptors in intestinal lumen and induce physiological reactions Some peptides are absorbed intact by a specific peptide transporter system into the circulatory system and transported to target organs

40. Responses to Feeding Functional Proteins or Peptides Antimicrobial – including control of gut microflora Antiviral Binding of enterotoxins Anti-carcinogenic Immunomodulation Anti-oxidant effects Opioid effects Enhance tissue development or function Anti-inflammatory Appetite regulation Anti-hypertensive Anti-thrombic

41. Functional Activity of Major Milk Proteins Caseins (α, β and κ) Transport of minerals and trace elements (Ca, PO4, Fe, Zn, Cu), precursor of bioactive peptides, immunomodulation (hydrolysates/peptides) β-Lactoglobulin Retinol carrier, binding fatty acids, potential antioxidant, precursor for bioactive peptides α-Lactalbumin Lactose synthesis in mammary gland, Ca carrier, immunomodulation, anticarcinogenic, precursor for bioactive peptides Immunoglobulins Specific immune protection (antibodies and complement system), G, M, A potential precursor for bioactive peptides Glycomacropeptide Antiviral, antithrombotic, bifidogenic, gastric regulation Lactoferrin Antimicrobial, antioxidative, anticarcinogenic, anti-inflammatory, immunomodulation, iron transport, cell growth regulation, precursor for bioactive peptides Lactoperoxidase Antimicrobial, synergistic effect with Igs and LF Lysozyme Antimicrobial, synergistic effect with Igs and LF Serum albumin Precursor for bioactive peptides Proteose peptones Potential mineral carrier

42. Functional Activity of Minor Milk Proteins Growth factors (IgF, TGF, EGF) stimulation of cell proliferation and differentation Cytokines regulation of immune system (interferons, interleukins, TGFβ, TNFα) Inflammation Increases immune response Milk basic protein (MBP) Promotion of bone formation and suppression of bone resorption Osteopontin Modulation of trophoblastic cell migration

43. Protein Fragments That Have Biological Activity

44. Functional Protein Effects During Toxin or Disease Challenge During intestinal inflammation, some functional proteins: Reduce local inflammatory response excessive activation of inflammatory cells permeability Increase Nutrient absorption Barrier function Intestinal health During intestinal inflammation, some functional proteins: Are absorbed and create adverse allergenic and immune responses in the body Modified from Campbell, 2007