‘Let’s talk about
Fibre Degrading Enzymes Natalia Soares Global Product Manager
Fibre degradation concepts
1. “our” fibre is of high molecular complexity
2. “our” Arabinoxylan looks like this:
3. And “a” is different from “a”
4. The substrate
(Knudson, 1997)
5. NSPase’s mode of action Release of nutrients
Viscosity reduc<on
Par<al degrada<on of insoluble NSP, resul<ng in the release of nutrients trapped in the cell wall fibre network A d d i < o n a l n u t r i e n t s available for the animal
Breakdown soluble structural NSP will result in digesta viscosity decrease Will avoid: reduced digesta transit impaired nutrient absorp<on bile acids deconjuga<on disbacteriosis wet liPer
6. Not all xylanases are the same Bacillus xylanase
A. aculeatus xylanase
Trichoderma xylanase
INSOLUBLE AX
SOLUBLE AX
not viscous
viscous !
not viscous
viscous !
viscous !
SUGARS & OLIGOSACCHARIDES not viscous
not viscous
7. Substrate specificity
(Moers et al (2003) A screening method for endo-‐β-‐1,4-‐xylanase substrate selec<vity)
Not all xylanases are the same!
8. Substrate variability
9. Enzymatic complex versus single activity
All xylanases are different!
Fibre is of high molecular complexity
Enzymes are selecEve: “a” is different from “a”
Substrate is highly variable
Specific degradaEon profile: (A)XOS?
Feeding the animal versus feeding microflora
Tailored efficiency â&#x20AC;&#x201C; enzymatic complexes
Baseline...
1 2 3
EnzymaEc complex
Non GMO T. citrinoviride with specific metabolic potenEal for enzyme producEon.
Unique combinaEon of enzyme acEviEes
Surface fermentaEon process – wheat bran substrate and “perfect by nature” complex of enzymes
OpEmal mode of acEon
Balanced hydrolysis of soluble and insoluble AX – viscosity reducEon and release of nutrients
Surface fermentation substrate
(Knudson, Â 1997) Â
Unique combination of activities ü
Creating UNIQUE enzymatic complexes: ü ü
ü
Trichoderma grows under stress: high temperature and moisture Non GMO Trichoderma growing over wheat bran: will produce the perfect enzymatic complex to degrade the substrate and use nutrients for its growth Under Surface Fermentation conditions there are metabolic pathways that are activated, that are not used when in submerged fermentation processes – so called fungi physiology of the solid medium. Meaning, there are enzymes that are only produced under SF
Targeted efficacy - developments
Enzyme activity on arabinoxylan Proportions of degradation products (HPSEC-RI)
Wageningen 2016
New research insights… • There is an op<mal fibre degrada<on • All enzymes are different • AXOS story is real (supports pigs mortality reduc<on story and addi<onal effects over fibre degrada<on and nutrient diges<bility DTT
Mw (kDa) 250
-‐
DTT+
Mw (kDa)
37
250 150 100 75 50 37
25 20
25 20
15
15
150 100 75 50
10
10 HX
Marke r
HC
Marke r
XB
XL
XLA XT
Marke r
Marke r
HX
HC
Marke r
XB
XL
XLA
XT
Marke r
XT XLA XL XB
Substituted AXOs
HX min 2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
24.0
26.0
28.0
30.0
32.0
34.0
36.0
38.0
40.0
Degradation profile of wheat AIS, WUS, WES Double -‐ subs<tuted
Single -‐ subs<tuted
DP2
DP4
Xylo-‐oligosaccharides (XOs) DP3
W-AIS
Glucose Xylose
AXOs
Mul<ple -‐ subs<tuted
HX, 24h Blank
W-WUS HX, 24h Blank
W-WES HX, 24h Blank Arabinoxylan oligosaccharides (AXOs) DP 8
DP 2 DP 3
DP 4
DP 4 Xylo-‐oligosaccharides (XOs)
Single-‐subs<tuted AXOs
DP 6 Double -‐subs<tuted AXOs
Mul<ple -‐subs<tuted AXOs
Linking research with animal performance • Broilers
Effect (%) of NSP degrading enzymatic complex supplementation on technical performance for various broiler diets (1500 EPU/kg) 4,00 3,00 2,00 1,00 0,00 -‐1,00 -‐2,00 -‐3,00 -‐4,00
extra growth broilers % improvement over control
2,85
improved FCR broilers (adj. for 2250 g) -‐3,10
21
Energy upliV (kcal ME/kg feed) with 1500 EPU/kg feed 25
0
Increase in metabolisable energy (kcal/kg feed)
200 175
150
125
100
75
50
140
120
100 60
40
120
104 94 111
89
80 33
20 0
Energy uplift by NSP degrading enzymatic complex (broiler diets)
22
Effect of Hostazym® X on gut micro flora of broilers Trial 1
control
Hostazym® X
BWG, g (42d) FCR
2745 1.59
2796 1.58
Total count (log cfu/ml) Lactobacilli count (log cfu/ml)
9.15
9.22
8.42
8.53
6.37a
5.85b
Caeca pH Trial 2
control
Hostazym® X
BWG, g (42d) FCR
2661 1.56
2683 1.55
Total count (log cfu/ml) Lactobacilli count (log cfu/ml)
9.21
9.56
8.50
8.79
6.32a
5.93b
Caeca pH
Microflora shifts? Distribution of microbial population in fecal samples from birds with low FCR
0,46
Distribution of microbial population in fecal samples from birds with high FCR
0,05
Campylobacteraceae Enterobacteriaceae
15,58
Campylobacteraceae
12,16
Enterobacteriaceae
17,29
Flavobacteriaceae
Flavobacteriaceae 0,55
Fusobacteriaceae
2,85
0,07
Moraxellaceae 46,96
Ruminococcaceae Xanthomonadaceae
Singh et al 2012
8,89
1,31
Fusobacteriaceae Moraxellaceae
10,02
8,06 8,2
Ruminococcaceae Xanthomonadaceae
Detection of pentose oligomers in ileal digesta § Performance Broiler trial – Warmia, PL
Sample
Treatment
T1
Control
T2
Hostazym X at 1500 EPU/kg
T3
Hostazym C low dose
T4
Prototype 1
T5
Prototype 2
T6
Prototype 3
T7
Prototype 4
Detection of pentose oligomers in purified ileal digesta samples (broilers 25d) P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17
T1
T5
T2
T6
T3
T7
400
T4
400
600
P11 P12 P13 P14 P15 P16
800
1000
1200
1400
1600
1800
2000
2200
2400 m/z
600
800
1000
1200
1400
1600
1800
2000
2200
2400 m/z
§ Arabinoxylan or xylan oligosaccharides up to DP 17 § Detected in T2, T4, T5, T6 and T7 § Were not detected in T1 (blank) or T3
Research project conclusions ü
Tested enzymatic complex HX is highly active on arabinoxylan, β-glucan and xyloglucan ü
ü
ü
ü
Endo-β-1,3-glucanase activity present!
HX complex could degrade soluble and insoluble fibre from wheat
(Arabino)xylan oligosaccharides were detected in ileal digesta from broilers fed with HX à in vivo formation of AXOs by Huvepharma enzymes confirmed AXOs were formed mainly due to the hydrolysis of soluble AX, while XOs were formed from insoluble AX à significant for prebiotic activity model
Conclusion Enzyma<c complexes show higher efficacy on degrading NSPs resul<ng in improved animal performance
NSP degrading enzyma<c complexes have beneficial effects beyond fibre degrada<on and technical performance contribu<ng for improved gut health status It is possible to tailor enzyma<c complexes for efficiency, suppor<ng the op<mal fibre degrada<on concept and allowing more flexible feed formula<on: high economic return without compromise zootechnical performance