Some enzymes work against anti-nutritional factors (ANF), which are produced by plants, in order to obtain more nutrients from plants for increased animal nutrition. The correct enzyme (or combination of enzymes) need to be used for the specific target. This depends on the plant cell wall structure. For example, the cell walls of the endosperm of corn is comprised mainly of insloluble arabinoxylans, which means that only xylanases are able to degrade this wall to increase the energy value of corn based diets. Read the presentation to find out more about the activity of different types of enzymes working alone or together.

1. How to overcome anti-nutritional factors
by using a range of enzymes
Global Feed and Food Congress 2013
South-Africa
April 10, 2013
J-P. Ruckebusch, I. Knap, M. Umar Faruk and E. Upton Augustsson

2. CONFIDENTIAL
Public/How to overcome anti-nutritional factors with a range of feed enzymes/J-P Ruckebusch/April, 2013
Anti-Nutritional Factors & Enzymes
• Plants produce ANFs as part of their normal metabolism
• ANFs interfere with animal’s ability to digest plant nutrients, thereby
reducing digestive efficiency by
• Binding to various nutrients
• Increasing viscosity of intestinal contents
• Inhibiting/deactivating endogenous digestive enzymes
• Irritating the intestinal mucosa (challenge to immune system)
• Specific mono-component enzymes work against ANFs by hydrolyzing:
• Phytin
• Non-soluble and soluble NSPs
• Trypsin inhibitors and lectin
• Other ANFs (ß-mannan, etc…)
Page 1
Source: Kumar, 1994; Choct et al., 1991; Lange et al., 2000; Cowieson 2004

3. Phytate life cycle
Proteins
Proteins
Proteins
Proteins
Phytate in
plant cell wall
Proteins
Phytate negative
charges bind
various positively
charged molecules
Proteins
Starch
Starch
Starch
Insoluble tertiary
protein-mineral-phytate
complexes with Ca
Page 2
Insoluble binary
protein-phytate
complexes
Source: Rajendran and Prakash, 1993; Kies et al., 2006; Prattley et al., 1982;
Montagne et al., 2001
Mineral-phytate
complexes

4. Phytate & Phytase in vivo
Cu++ -O
O
-O
Ca++
-O
P
O
-O
O
O
O
CH
K + -O
P
Na+
HC
O
O
- Available Ca
Less
excretion
Bone strength
and
mineralisation
O
Zn++
Physiologic
functions
- Available minerals
- Available amino acids*
Page 3
O-
P
O-
Various soluble IP3 are
hydrolyzed by
endogenous enzymes
Hydrolyses IP6 into IP3
Liberates P Ca, Zn, …
,
Reduces protein binding
- Available P
O- Mg++
O
P
-O
Fe++
O-
O
C
H
HC
O
-O
P
CH
H
C
O
O-
P
Source: Mroz, Jongbloed et al., 1994; Rapp et al., 2001; Kemme et al., 2006
*48 PR papers, 34 in poultry, 80% positive effects on AA digestibility
Growth
performances

5. Phytate w/o Phytase in vivo
In absence of phytase, phytates bind
with proteins into insoluble
macromolecular aggregates
Protein degradation into
peptides by pepsin is reduced
Peptide production
regulation is disturbed
w gastric hyper-secretions
of HCl and pepsin
Na conc. increases
reducing Na dependent
transport
Decreased intestinal
uptake of dietary and
endogenous amino acids
Page 4
Increased acidity of
duodenal content leads to
increased production of
mucin and Na-HCO3

6. Phytate w Phytase in vivo
In presence of phytase, phytates (IP6)
are hydrolyzed in smaller phytate
esters like IP4, IP3…
Small phytate esters are
soluble and do not bind
proteins. They will be
degraded and absorbed
Not bound proteins are
available for hydrolysis
into peptides and amino
acids
By indirect action
phytase increases nutrient
digestibility
Page 5

7. Model of plant cell wall
A mixture of polysaccharides interlocked in a complex structure
Xyloglucan network
Cellulose fibrils
Cellulose fibrils
Glucuronoarabinoxylan
Monocotyledonous plants
(like corn, wheat, barley)
Page 6
Pectic polymer matrix
Dicotyledonous plants
(like soya, canola, sunflower)
Source: Carpita, N.C. and Gibeaut, D. M (1993).The Plant Journal 3(1):1-30.

8. Different plant cell walls’ carbohydrates
Different plant cell wall soluble and insoluble carbohydrates
require different enzymes solutions
Page 7
Source: Bach Knudsen KE., 1997; Choct. M.,1997

9. The insoluble cell wall structure in corn
Endosperm cell wall w.
insoluble arabinoxylans
Broken down by xylanase
Releasing cell contents
The thin cell walls can be described as
arabinoxylan cages encapsulating nutrients.
Page 8
Source: Evers and Millar 2002

10. Degradation of cell walls by xylanase
0 min
Wheat cuts right after injection of xylanase
180 min
Wheat cuts 180 minutes after
treatment with xylanase
Fluorescence is induced by UV irradiation of plant materials,
then emitted by mainly ferulic acid cross-linking arabinoxylan chains.
Page 9
Source: Le et al., 2013

11. Degradation of cell walls by xylanase
0 min
Corn from piglets digesta sample
right after injection of xylanase
120 min
Corn from piglets digesta sample
120 minutes after treatment with xylanase
The degradation of cell walls by a xylanase is monitored indirectly
as a decrease in fluorescence resulting from the breakdown of the
arabinoxylan rich cell walls into soluble oligomers.
Page 10
Source: Novozymes, unpublished

12. Public/How to overcome anti-nutritional factors with a range of feed enzymes/J-P Ruckebusch/April, 2013
Degradation of cell walls by pure xylanase
Xylanase hydrolyses the
non-soluble fraction
of plant cell walls
Xylanase hydrolyses the
soluble fraction of plant cell walls
Decreases
intestinal viscosity
Opens the cell
wall structure
Releases the
entrapped
nutrients
Increases passage
rate of digesta
Give access to
digestive enzymes
to cell content
Increases
Feed Intake
Xylanase
Xylanase
Insoluble
arabinoxylans
Page 11
Reduces substrate for
negative microflora
Viscous soluble
arabinoxylans
Non viscous soluble
arabinoxylans

13. Different mode of action of pure xylanases
The in vivo confirmation
Active on both
soluble and
insoluble
arabinoxylans
2,156
Weight gain (g/bird/week)
390
Active only on 2,2
soluble arabinoxylans
2,15
380
2,1
2,035
370
2,05
2,005
360
2
1,965
350
1,95
340
1,9
330
345,4
392,2
394,8
382,9
1,85
320
Control
Xylanase
Exp. xylanase
Best performances require xylanase activity
against both insoluble and soluble xylans.
Page 12
Source: Choct, M., Kocher, A., Waters, D.L.E., Pettersson, D. and Ross, G., 2004
Test product
FCR
400
Active only
on insoluble
arabinoxylans

14. CONFIDENTIAL
Feed Protease degrades soya ANFs
Kunitz Inhibitor
Lectin
Bowman Birk Inhibitor
Degradation % Protease Trypsin
Lectin
11
Kunitz
96
4
Bowman Birk
Page 13
98
64
29
Source: Nielsen et al., 2013

15. CONFIDENTIAL
ANFs in soy and protease’s efficacy
Protein hydrolysis - ABS 340 nm
Trypsin
Feed Protease
Feed Protease, 1000 Units/kg SBM
Pancreas Trypsin Novo, g/kg SBM
Raw soy
SBM 1
SBM 2
Feed Protease acts by reducing Lectin level and KTI and BBI levels, thereby
reducing the sensitivity of animal performance to low quality soybean meal.
Page 14
Source: Nielsen et al., 2013

16. CONFIDENTIAL
ANFs in soy and protease’s efficacy
Protein hydrolysis - ABS 340 nm
Pancreatic
Enzymes +
Feed Protease
Pancreatic
Enzymes
Feed Protease
Commercial dose
Pancreas Trypsin Novo, g/kg SBM
The addition of the pure feed protease completes and doubles the
efficacy of endogenous pancreatic proteases (trypsin and chymotrypsin).
Page 15
Source: Nielsen et al., 2013

17. CONFIDENTIAL
Raw soybean & protease on broilers
Body weight gain (g/b)
1200
1000
+4% (P<0.05)
+2% (P>0.05)
1092
1141
+3% (P<0.05)
1016
1037
867
800
897
600
400
200
0
Page 16
0
15000
0% RSB
Source: Umar Faruk et al., 2012
0
15000
10% RSB
0
15000
20% RSB

18. CONFIDENTIAL
Raw soybean & protease on broilers
Body weight gain (g/b)
1200
1000
+4% (P<0.05)
+2% (P>0.05)
1092
1141
+3% (P<0.05)
1016
1037
867
800
897
600
400
200
0
Page 17
0
15000
0% RSB
Source: Umar Faruk et al., 2012
0
15000
10% RSB
0
15000
20% RSB

19. CONFIDENTIAL
Feed Protease combined with phytases
Degree of protein hydrolysis
+/- protease
140
100
80
%60
n=4
n=4
40
20
P-release from InsP6 reletive to
%
’phytase alone’ (%)
DH relative to ’no protease’ %
110
*
120
0
No protease
P-release from phytate (IP6)
+/- protease
100
90
80
70
60
50
Protease
* Significantly different from control
(All-pairs Tukey Kramer HSD, P<0.05)
Overall significant positive effect of phytase+protease
compared to phytase alone on phytate degradation
Page 18
Source: Pontoppidan et al., 2012

20. CONFIDENTIAL
Feed Protease combined with xylanases
Degree of protein hydrolysis
+/- protease
120
*
250
Xylose solubilization
+/- protease
100
200
150
%
n=9
100
50
n = 15
0
No protease
Protease
Soluble xylose relative
to ’xylanase alone’ %
(%)
DH relative to ’no protease’ %
300
80
60
40
20
0
* Significantly different from control
(All-pairs Tukey Kramer HSD, P<0.05)
Overall significant positive effect of xylanase+protease
compared to xylanase alone on xylose degradation
Page 19
Source: Pontoppidan et al., 2012

21. CONFIDENTIAL
One Substrate, One enzyme
• Studies on ANFs in feed ingredients on which mono-component enzymes
have proven to be effective provide a better understanding of the mode
of action of specific feed enzymes.
• It also helps in predicting the nutritional contribution a single enzyme or
a combination of enzymes can make to the value of a diet.
• Enzymes are highly specific. The non-soluble and soluble NSPs are more
complex substrates and may require more than one enzyme to alleviate
the ANF’s impact.
• The choice of commercial feed enzymes and combination must be made
based on the best knowledge available, not only on the enzyme activity
itself, but on the substrate it is targeting.
Page 20

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