Protein Digestion

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

H3N+ C

HC

R

O

NH

CH

CO

RNH

CH

C

R

O

O–

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

Aromatic amino acids

Pepsinogen

HClPepsin

Protein Digestion – Small Intestine

Pancreatic enzymes secreted Trypsinogen Chymotrypsinogen Procarboxypeptidase Proelastase Collagenase

Zymogens

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

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

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

Protein Digestion Proteins are broken down to

Tripeptides Dipeptides Free amino acids

Free Amino Acid Absorption

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+

Amino Acid Transporters – Brush Border Membrane

Transport system

Energy required

Substrates carried

LBIMINOy+

Bo,+

bo,+

NoYesYesNoYesNo

Leu, other neutralPhe, Tyr, Trp, Ile, Leu,

ValPro, Gly

Basic amino acidsMost neutral and basicMost neutral and basic

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

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)

In the Enterocytes…

First cells that can use the amino acids Transport into portal

blood Protein synthesis

Digestive enzymes Structure and growth

Energy

Stoll et al. (1998)

%

Groff & Gropper, 2000

*Whole proteins are nutritionally insignificant...

Basolateral Membrane Transport of

free amino acids only* Peptides are

hydrolyzed within the enterocyte

Transport mainly by diffusion and Na-independent carriers

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

Groff & Gropper, 2000

Overview of Protein Digestion and Absorption in Monogastrics

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

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

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

Protein Degradation in RumenFeedstuff % Degraded

in 2 hours

Urea 100

Alfalfa (fresh) 90

Wheat Grain 78

Soybean Meal 65

Corn Grain 48

Blood Meal 18

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)

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

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!)

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)

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

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)

Microbial Protein Synthesis Synchronization of carbohydrate and N availability

NPN supplementation Carbohydrates used for carbon skeleton of amino acids

VFA (CHO fermentation)

Rumen NH3

Blood NH3

Adapted from Van Soest, 1994

Time post-feeding

Con

cent

ratio

n

Carbon backbone(from CHO fermentation)

Microbial Protein Formation

Dietary NPN

Dietary Soluble RDP

Microbial ProteinsAmino

Acids

Carbon Skeletons

Sulfur

Other Co-factors

NH3 ATP

Dietary Starch Sugar

Dietary Cellulose Hemicellulose

rapid

slow

rapid

slower

Dietary Insoluble RDP

very slow

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

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

Urea

Ammonia + CO2

Urease

Feed Protein, NPN and CHO

Feed Protein

Feed NPN

NH3/NH4

Bacterial N

NH4+ loss

MCP

RDP

RUPFeed Protein

AA

MCP

AA

NH3

Liver

Blood Urea

Salivary N

ATP

RUMEN

SMALL INTESTINE

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

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

Salivary Urea

NPN

NH3

POOL

Dietary Nitrogen Non-utili

zed Ammonia

NH3 UREA

LIVER

LEVEL TOPROVIDE FORMAXIMUMMICROBIAL GROWTH

MICROBIAL PROTEIN

65% OF PROTEIN

35% OF PROTEIN

SMALL INTESTINE

AMINO ACIDS

AMINO ACIDSPROTEIN

AMINO ACIDS

PEPTIDES

Reticulo-rumen

RUP

RDP

Recycled urea

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

Functional Proteins

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

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

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

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

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

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

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

Protein Fragments That Have Biological Activity

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