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+ CH

C

R

O

NH

CH

C

O

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 HCl

Pepsin

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

L

B

IMINO

y+

Bo,+

bo,+

No

Yes

Yes

No

Yes

No

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

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 Rumen

Feedstuff % 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

Concentr

ation

Carbon backbone (from CHO fermentation)

Microbial Protein Formation

Dietary

NPN

Dietary

Soluble RDP

Microbial

Proteins

Amino 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

RUP Feed

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

NH3 UREA

LIVER

LEVEL TO

PROVIDE FOR

MAXIMUM

MICROBIAL GROWTH

MICROBIAL PROTEIN

35% OF PROTEIN

SMALL INTESTINE

AMINO ACIDS

AMINO ACIDS PROTEIN

AMINO ACIDS

PEPTIDES

Reticulo-rumen

RUP

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