Download What more do we need to know to optimize the

What more do we need to know to
optimize the use of a protease?
Roselina Angel
Department of Animal and Avian Sciences
University of Maryland
College Park, MD, USA
Road map
• Lessons from the field
• May help us understand limits to protein digestion
• Possible areas for enhancing protease efficacy
• Why do we see the effects that we see?
•
•
•
•
Stepwise digestion of protein
Proteins resistant to digestion
Importance of pH and passage rate
Endogenous losses and sources of undigested protein
• Nutrient-enzyme interactions
• Impact of a protease on ingredient AA digestibility
Impact of water pH and age on intestinal
segment pH and nutrient digestibility
Effect of water pH (5.8) on pH in intestinal segments
Jejunum
8 d - 6.4
38 d - 6.4
Ileum
8 d - 6.6
38 d - 6.8
Ceca
8 d - 6.7
38 d - 6.1
Crop
8 d - 6.0
38 d - 6.1
Proventriculus
8 d - 1.9
38 d - 1.4
Gizzard
8 d - 2.9
38 d - 2.2
Duodenum
8 d - 5.8
38 d - 5.5
Angel et al., 2013- 6 replicates of 3 or 4
broilers/rep, at 8 and 38 d of age
Effect of water pH (5.8 and 8.1) on pH in intestinal segments
Jejunum
8 d 6.4/6.6
38 d 6.4/6.5
Ileum
8 d 6.6/7.1
38 d 6.8//7.0
Ceca
8 d 6.7/7.3
38 d 6.1/6.9
Crop
8 d - 6.0/7.6
38 d - 6.1/7.5
Proventriculus
8 d 1.9/4.1
38 d 1.4/2.9
Gizzard
8 d 2.9/5.6
38 d 2.2/4.6
Duodenum
8 d 5.8/6.0
38 d 5.5/5.7
Angel et al., 2013- 6 replicates of 3 or 4
broilers/rep, at 8 and 38 d of age
Effect of water pH on nutrient digestibility
Jejunum
8 d - 6.4/6.6
38 d - 6.4/6.5
Crop
8 d - 6.0/7.6
38 d - 6.1/7.5
Proventriculus
8 d - 1.9/4.1
38 d - 1.4/2.9
Gizzard
8 d - 2.9/5.6
38 d - 2.2/4.6 Duodenum
8 d - 5.8/6.0
38 d - 5.5/5.7
(Angel et al., 2013)
Ileum
8 d - 6.6/7.1
38 d - 6.8//7.0
Ceca
8 d - 6.7/7.33
38 d - 6.1/6.9
Apparent ileal DM digestibility
84
85.1 a
5.8 pH
8.1 pH
82.3
82
a
%
80
78.7
78
76
73.9
74
N =6 , 3 or 4 broilers/rep
at 9 and 38 d of age,
respectively
c
72
SEM 2.3
8d
Age
38 d
b
Effect of water pH on nutrient digestibility
Ileum
8 d - 6.6/7.1
38 d - 6.8//7.0
Jejunum
8 d - 6.4/6.6
38 d - 6.4/6.5
Crop
8 d - 6.0/7.6
38 d - 6.1/7.5
(Angel et al., 2013)
Ceca
8 d - 6.7/7.33
38 d - 6.1/6.9
Proventriculus
8 d - 1.9/4.1
38 d - 1.4/2.9
Gizzard
8 d - 2.9/5.6
38 d - 2.2/4.6
90
Duodenum
8 d - 5.8/6.0
38 d - 5.5/5.7
Apparent ileal protein digestibility
5.8 pH
8.1 pH
85.1
a
80
%
80.1
74.2 c
70
60
55.2 d
N =6 , 3 or 4 broilers/rep at 9
and 38 d of age, respectively
50
SEM 2.1
8d
Age
38 d
b
STEPS in protein digestion
• Gastric area: Acid unfolding/Pepsin digestion
– Polypeptides, oligopeptides
• Intestinal luminal: pancreatic enzymes (trypsin,
chymotrypsin, elastase, carboxypeptidases)
– Oligopeptides (2 to 6 aa), free amino acids
• SI Mucosal cells – peptidases (membrane bound)
– Di and tri peptides, free amino acids
• Intraluminal – cytosolic peptidases
– Free AA
Role of gastric acidity
• Denature (unfold) proteins to allow access for
enzymes to hydrolyz
• Extent of protein hydrolysis in gizzard
correlated to time of exposure to pepsin/HCl
(Keller, 1968)
• Any inhibitions to this first step will partially
inhibit subsequent steps
– Type of protein
– Acidity in gastric area
Resistant protein in soybeans
(Wang et al., 1995)
• High molecular fraction (HMF) resulting from in
vitro digestion of soybean protein with pepsin
and pancreatin – when fed to rats
• Resistant to further digestion – high homology
between AA pattern of HMF and AA in feces
• Bound to bile acids results in increased bile acid
secretion
• Increased cholesterol excretion
• Increased N and fat excretion
Proteins resistant to digestion
(refractory)
• Soybean proteins
– Glycinin – accounts for 50+ % of SB proteins
• Varies with cultivar
– High in disulfide groups
– 5 subunit polypeptides tightly bound by disulfide
bridges
Proteins resistant to digestion
• Soybean proteins – Glycinin
– 10.3% of glycinin (portions) fed to pre ruminant
calves was still present in the distal ileum (Tukur et
al., 1993)
– From heat treated soy flour (for milk replacers)
• Implications
– In an 18% protein poultry diet containing 20% SBM
(48% protein)
– Would result in a 2.5 to 3% reduction in protein
digestibility
Proteins resistant to digestion
• Sunflower
– 2S albumin proteins – high in cystine
• 13 subunits
• SFA8 subunit highest in sulfur AA
• Both SFA8 and LTP subunits bind lipids
– Both subunits resist in vitro digestion (Berecz et
al., 2013)
• Association with lipids decreases lipid digestion
• Quantification has not been done
Proteins resistant to digestion
• Wheat
– Gliadin (and related proteins from rye and barley)
very rich in proline
• 13 subunits
• SFA8 subunit highest in sulfur AA
• Both SFA8 and LTP subunits bind lipids
– Proline rich residue difficult to digest (Hausch et
al., 2002)
• Supplementation of a prolyl-edopepdidase improved
digestibility of proline rich olygopeptides
Effect of water pH intestinal pH
Crop
8 d - 6.0/7.6
38 d - 6.1/7.5
(Angel et al., 2013)
Ileum
Impact of pH
pepsin activity and stability
8 d - on
6.6/7.1
Jejunum
8 d - 6.4/6.6
38 d - 6.4/6.5
38 d - 6.8//7.0
(From
Piper and Fentone, 1965)
Ceca
8 d - 6.7/7.33
38 d - 6.1/6.9
Proventriculus
8 d - 1.9/4.1
38 d - 1.4/2.9
Gizzard
8 d - 2.9/5.6
38 d - 2.2/4.6 Duodenum
8 d - 5.8/6.0
38 d - 5.5/5.7
N =6 , 3 or 4 broilers/rep at 9
and 38 d of age, respectively
Effect of age on pH of the proventriculus
Bowen and Waldroup, 1969
Jimenez-Moreno et al., 2010
From Rynsberger, 2009, Mean of 10 b/age
Impact of diet Ca and age on gizzard content pH
(Corn-SBM St diet, no added inorganic P)
pH at 5 d of age
pH at 32 d of age
5.0
5.0
y = 1.6649x + 2.6552
R² = 0.7071
4.5
4.0
4.5
3.5
3.5
3.0
3.0
2.5
2.5
2.0
2.0
1.5
1.5
SEM 0.042
1.0
0
0.2
0.4
y = 1.1893x + 1.7993
R² = 0.447
4.0
0.6
tP = 0.42%, PP = 0.30%, nPP = 0.12%
Limestone added
Analyzed Ca 0.19 to 0.95
0.8
SEM 0.068
1.0
1
0
0.5
n=8
Angel et al., Unpublished
1
Impact of age on protein digestibility
Protein digestibility, chickens
95
%
90
85
Batal and Parsons, 2002
Chickens, corn-SBM diet
80
75
4d
7d
10 d
14 d
21 d
Noy and Sklan, 1997; Corn-SBM diet
WHY?
Sites of protein digestion and absorption
Net Digestion and absorption
Hurwitz et al., 1972
• Effect of dietary Ascaridia galli infection (most
parasites in upper SI)
• Effect of SI segment
• SBM/Milo diet
• 20.4 vs 14.4 % protein diets
• White leghorn males– Infected at 1 week of age
• Fed experimental diets for 5 days, starting at 5
weeks of age
Hurwitz et al, 1972
Upper Jejunal digestion
No infection @ 60%
Infected
@60%
Duodenal digestion
Non infected @ 8%
Infected
@50%
Non infected
Infected with Ascardia galli
Upper Jejunal absorption
No infection @ 40%
Infected
@40%
Duodenal absorption
Non infected @ -7%
Infected
@ -57%
AA or peptide absorption
• Different mechanisms of absorption
• High competition for absorption of free AA
– 5 transporter systems (Broer, 2008)
– Transporter systems mostly Na dependent
• More efficient and more rapid absorption of
peptides
Sources of protein entering the
intestine
Protein in the GIT – origin – feed or endogenous?
How much and where digested?
Pigs (from Fuller and Reeds, 1998)
Feed N –
Endogemous N –
35.3 grams/day
16.1 grams per day
Stomach
Endogenous N secretions
g/d
Feed N
5.3
N entering the GIT = 2.2 g feed N: 1 g endogenous N
Jejunum
8.9
Ileum
Large Intest.
1.9
Undigested
2.6 (7.4%)
35.3
1.6 (10%)
Absorbed N
Feed
28.2 (80%)
4.3 (12.6%)
Endogenous
10.4 (65%)
4.1 (25%)
Basal endogenous losses
• Diet Independent endogenous flow decreases with age, chicks
All AA -g/kg of DM intake
Adedokun et al., 2007
Endogenous losses with or without a protease
mg of AA/ kg DM Feed intake
800
Mateos et al., 2014
700
Mateos et al., 2014 +
* * *
600
500
400
300
200
100
Thr
Val
Met
Cys
Lys
Iso
Gly
Pro
Rate of passage
pH and mean retention time digesta in the GIT broilers 22 d of age
(mean 25 broilers, min/max) (Angel et al., 2013)
Mean feed particle size 0.822 mm (corn/SBM diet)
Jejunum
6.1 (5.8/6.6)
Ileum 6.5 (6.2/7.1)
Ceca
6.4 (5.9/6.9)
Crop
5.2 (4.3/5.9)
Large intestine
6.4 (6.0/7.3)
5.0 (4.5-5.4)
Proventriculus
1.6 (1.15/2.46)
6.3 (5.9-6.6)
Gizzard
2.5 (1.6/3.2)
Duodenum
6.0 (5.5/6.4)
Total residence time as affected by age
10 d = 3h 15 min (2:32 -3:51 ) (SEM:09)
22 d = 4 h 25 min (3:10 – 4:42) (SEM:14)
30 d = 4 h 44 min (3:30 – 5:32) (SEM:19
42 d = 5 h 10 min (4:09-6:05) (SEM:25)
pH and mean retention time digesta in the GIT broilers 22 d of age
(mean 25 broilers, min/max) (Angel et al.,2013)
Mean feed particle size 0.822 mm (corn/SBM diet)
Jejunum
6.1 (5.8/6.6)
D+J = 87 min
Crop
5.2 (4.3/5.9)
12 min
Ileum 6.5 (6.2/7.1)
80 min
Ceca
6.4 (5.9/6.9)
Large intestine
6.4 (6.0/7.3)
Proventriculus
1.6 (1.15/2.46)
37
min
)
Gizzard
2.5 (1.6/3.2)
Total residence time as affected by age
Duodenum
6.0 (5.5/6.4)
10 d
22 d
30 d
42 d
= 3h 15 min (2:32 -3:51 ) (SEM:09)
= 4 h 25 min (3:10 – 4:42) (SEM:14)
= 4 h 44 min (3:30 – 5:32) (SEM:19
= 5 h 10 min (4:09-6:05) (SEM:25)
Retrograde movement of digesta
• Ocurrs in 3 areas (Duke, 1994)
– a) Between the proventriculus and gizzard
– b) From the ileum/jejunum through the duodenum
the gizard/proventriculus
– c) From the cloaca to ceca and proximal ileum
Retrograde movement of digesta (Sklan et al., 1978)
• Injected a radioactive isotope into bile ducts
– Within 2 min – of injected dose
– 40% in gizzard (50% still there after 20 min)
– 30% in duodenum
• Injected into upper Jejunum
– 20% in gizzard within 2 min
• Trypsin and lipase found in gizzard content
– were still active when content incubated at 6.5 pH
Relationship of passage rate and
digestibility
• Correlation between slower passage rates and
increased nutrient digestibility poultry
(Amerah et al., 2007; Svihus, 2011)
• As passage rate slows, dry matter digestibility
increase in gestating sows (Kim et al., 2007)
• Fast emptying of the stomach resulted in
decreased protein digestibility in calves
(Toullec and Lalles, 1995; Lalles et al., 1999)
Endogenous enzymes and nutrient
interactions
• Effect of feeding raw or heated SBM (Sklan et al., 1975)
– Increases bile acid secretion 2X
90
• But partially reabsorbed in lower SI
• 40% greater excretion in raw SBM 85
%
fed chicks
80
– No effect on lipase activity at any
75
point in the SI
70
Free fatty acid net retention
Heated SBM
Raw SBM
P<0.05
Endogenous enzymes and Nutrient
interactions
• Effect of feeding raw or heated SBM (Sklan et al., 1975)
– Net absorption of fatty acids
• 87.0 vs 75.6%
• Complexation of FFA with undigested protein
Triglycerides
(polypeptides/oligopeptides)
(Johnson, 1963; Sklan et al., 1975)
Free fatty
acids
(FFA)
• Importance of understanding nutrient interactions
Diglycerides
Monoglycerides
Phospholipids
Impact of ProAct on diet and
ingredient digestibilities
Reference method (apparent)
Bertichini et al 2009
Full fat soy vs SBM
% undigestible AA
45
%
40
35
30
25
20
15
10
5
0
-5
Full fat Soy
SBM
Improvement in essential apparent AA digestibility with 200 ppm
protease
12
% improvement
SBM
Full fat soy
10
8
6
4
2
0
Met
Lys
Thr
Cyst
Ile
Bertechini et al. (2009 )
Arg
Val
Phen
Leu
Soybean Meal
Nutrient
(analyzed)
Bertichini et al., 2009
Angel, et al., 2010
Fat (%)
3.59
2.88
Moisture (%)
10.74
8.8
C. Protein (%)
44.44
48.0
Urease, pH change
0.03
0.01
Solubility KOH, %
88.15
89.6 (87 to 94.2)
SBM- Amount of undigested AA (W/O ProAct)
45
40
% 35
30
25
20
15
10
5
0
Bertechini t al, 2009 Ref method (apparent)
Angel et al., 2010 NDF (Standardized)
SBM– percent of undigested – digested by ProAct
40
35
%
30
25
20
15
10
5
0
-5
Bertechini t al, 2009 Ref method (apparent)
Angel et al 2010, NDF (True)
SBM - Standardized ileal (SI) AA digestibility with 0 or 200
ppm of a commercial protease (Angel et al 2011)
92
Broiler
Turkey
Layer
% digestibility
87
82
77
72
Met
Lys
Thr
Cyst
Ile
Arg
Val
SBM – Improvement in SI AA digestibility with use of 200 ppm
of a commercial protease (Angel et al 2011)
% digestibility improvement
11
9
*
*
*
*
Broiler
Turkey
*
Layer
*
7
*
5
*
*
*
*
*
3
1
-1
-3
Met
Lys
Thr
Cys
Iso
Arg
Val
Try
How can we improve protein
digestion/absorption?
• What should be the target of an exogenous
protease?
• Unclear what the rate limiting step is in digestion
• Ingredient/diet related
• BUT there is information that allows us to
potentiate the effect of proteases
How can we improve protein digestion/absorption?
• BUT there is information that allows us to
potentiate the effect of proteases
– Look at water pH/diet buffering capacity
• Lowering pH, especially in young birds, can help with
protease efficacy
– Look at particle size, fiber concentration and type as means of decreasing gastric pH and reducing
passage rate
How can we improve protein digestion/absorption?
• BUT there is information that allows us to
potentiate the effect of proteases
– Look closely at what ingredients are in the diet
• Make sure NSP enzymes are present when needed –
impacts on digestibility of all other nutrients
• Use of phytases will increase the proteins available for
digestion – interactions with proteases?
• Make sure fat levels are not to low or to high – will
change passage rate, and potentially interact with other
digestive processes
How can we improve protein digestion/absorption?
• BUT there is information that allows us to
potentiate the effect of proteases
• Understand your ingredients
– Low digestibility nutrients – higher potential for
enzyme to work
– Apply meta analysis knowledge – What diets
(ingredients) and with what characteristics will have
the greatest potential for a protease to work on
– Diet Ca concentrations and solubility
– Refractory proteins in ingredients are potential feed
proteins that can be digested by exogenous proteases
How can we improve protein
digestion/absorption?
• BUT there is information that allows us to potentiate the
effect of proteases
• Understand the impact of diet (ingredient) on
endogenous losses
– Anti nutrient content (trypsin inhibitors, phytate, …), fiber, …
• Pay attention to AA balance – essential vs. non essential
amino acids
• Look for balance in aa and peptide residue for absorption
(not looking for all AA as a result of protein digestion)
How can we improve protein digestion/absorption?
• BUT there is information that allows us to
potentiate the effect of proteases
• Feed back mechanisms of nutrients on enzyme
secretion
• How does it change endogenous losses
• Costs of digestion
• What is really the N portion that is undigested
• Endogenous
• Decrease endogenous losses (fiber, anti nutrients,
exogenous enzymes, ..)
• Feed proteins – What feed proteins are poorly
digested?
Remember factors affecting digestion
• Microbial populations
• Host-diet-microbial population interactions
• Effect overall and intestinal health
• Affect how diets are digested, nutrients absorbed and
utilized
• More than enzymatic
•
•
•
•
Accessibility of substrate to enzymes (“caging”)
Interactions between nutrients (complexations)
Interaction between exogenous enzymes
Interactions between diet/endogenous enzymes/exogenous
enzymes
Thank you!
Questions?