Protein Digestion

Monogastric Protein Digestion

Whole proteins are not absorbed

Too large to pass through cell membranes intact O H

H3N+ C R C N H H C R O C N H H C R O C O

Digestive enzymes

Hydrolyze peptide bonds Prevents self-digestion

Secreted as inactive pre-enzymes

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 Pepsinogen

HCl

Pepsin

Cleaves at phenylalanine, tyrosine, tryptophan Aromatic amino acids

Protein leaves stomach as mix of insoluble protein, soluble protein, peptides and amino acids

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 Enteropeptidase/Trypsin Trypsin

Endopeptidase

Cleaves on carbonyl side of Lys & Arg

Chymotrypsinogen

Trypsin

Chymotrypsin

Endopeptidase

Cleaves carboxy terminal Phe, Tyr and Trp

Procarboxypeptidase

Trypsin

Carboxypeptidase

Exopeptidase

Removes carboxy terminal residues

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

Na+

Na+

Entrance of some AA is via active transport

Requires energy

Amino Acid Transporters Brush Border Membrane

Transport system L B IMINO y+ Bo,+ bo,+ Energy required No Yes Yes No Yes No Substrates carried Leu, other neutral Phe, Tyr, Trp, Ile, Leu, Val Pro, Gly Basic amino acids Most neutral and basic Most 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 Endocytosis Pinocytosis

Intracellular routes

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)

Basolateral Membrane

Transport of free amino acids only*

Transport mainly by diffusion and Na-independent carriers

Peptides are hydrolyzed within the enterocyte

Groff & Gropper, 2000

*Whole proteins are nutritionally insignificant...

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

Overview of Protein Digestion and Absorption in Monogastrics

Groff & Gropper, 2000

Ruminant Protein Digestion

Ruminants can exist with limited dietary protein sources due to microbial protein synthesis

Essential amino acids synthesized Rapid growth High production

Microbial protein is not sufficient during:

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 animals protein requirements

Escape protein, bypass protein, or rumen undegradable protein (RUP)

Aldehydes increase inter-protein cross-linking Heat treatment Digestibility of RUP source in the small intestine Protein quality

Utilization depends on

Protein Degradation in Rumen

Feedstuff
Urea Alfalfa (fresh)

% Degraded in 2 hours 100

90

Wheat Grain
Soybean Meal

78
65

Corn Grain
Blood Meal

48
18

Rumen Protein Utilization

Factors affecting ruminal degradation

Rate of passage

Rate of passage degradation

Solubility in water

Must be solubilized prior to degradation

Degradation

Heat treatment

N (and S) availability Energy availability (carbohydrates)

Protein Fractions

Dietary proteins classified based on solubility in the rumen

NPN, instantly solubilized/degraded Potentially degradable Insoluble, recovered in ADF, undegradable

B1 B2 B3

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

Microbes re-synthesize amino acids

Amino acids NH3, VFAs, and CO2

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

Available nitrogen

ATP

Available carbohydrates

NPN Degraded feed intake protein nitrogen (RDP) 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

Concentration

VFA (CHO fermentation) Blood NH3

Rumen NH3
Carbon backbone (from CHO fermentation)

Time post-feeding
Adapted from Van Soest, 1994

Microbial Protein Formation

Dietary Starch Sugar
rapid

Dietary Cellulose Hemicellulose

slow

Dietary NPN
rapid

Carbon Skeletons

Sulfur

Other Co-factors Microbial Proteins

NH3

ATP
Amino Acids
slower very slow

Dietary Soluble RDP

Dietary Insoluble RDP

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

Plasma urea enters rumen

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 Saliva Diffuses through rumen wall from blood

Urea

Urease

Ammonia + CO2

Feed Protein, NPN and CHO

RUP

Feed Protein NH3

AA

Feed RDP Protein

Feed NPN

NH3/NH4

SMALL INTESTINE
Bacterial N MCP MCP AA

Salivary N

NH4+ loss

RUMEN

Liver
ATP

Blood Urea

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

Recycled urea
NH3 UREA

LIVER NPN
Dietary Nitrogen

PEPTIDES
AMINO ACIDS

NH3 POOL

LEVEL TO PROVIDE FOR MAXIMUM MICROBIAL GROWTH

AMINO ACIDS
AMINO ACIDS

MICROBIAL PROTEIN

PROTEIN

SMALL INTESTINE
35% OF PROTEIN

RUP

Reticulo-rumen

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

Fermented animal-based products

Fish meal Meat and bone meal

Soy products

Yeast Lactobacillus organisms

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 (, and )

-Lactoglobulin

Transport of minerals and trace elements (Ca, PO4, Fe, Zn, Cu), precursor of bioactive peptides, immunomodulation (hydrolysates/peptides) Retinol carrier, binding fatty acids, potential antioxidant, precursor for bioactive peptides Lactose synthesis in mammary gland, Ca carrier, immunomodulation, anticarcinogenic, precursor for bioactive peptides Specific immune protection (antibodies and complement system), G, M, A potential precursor for bioactive peptides Antiviral, antithrombotic, bifidogenic, gastric regulation Antimicrobial, antioxidative, anticarcinogenic, anti-inflammatory, immunomodulation, iron transport, cell growth regulation, precursor for bioactive peptides Antimicrobial, synergistic effect with Igs and LF Antimicrobial, synergistic effect with Igs and LF Precursor for bioactive peptides Potential mineral carrier

-Lactalbumin

Immunoglobulins

Glycomacropeptide

Lactoferrin

Lactoperoxidase

Lysozyme

Serum albumin

Proteose peptones

Functional Activity of Minor Milk Proteins

Growth factors (IgF, TGF, EGF)

Cytokines

stimulation of cell proliferation and differentation regulation of immune system (interferons, interleukins, TGF, TNF)
Inflammation Increases immune response

Milk basic protein (MBP)

Osteopontin

Promotion of bone formation and suppression of bone resorption

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 Nutrient absorption Barrier function Intestinal health

Increase

During intestinal inflammation, some functional proteins:

Are absorbed and create adverse allergenic and immune responses in the body

Modified from Campbell, 2007