nutriFORUM 2019 - Ponencia sobre enzimas degradadoras de fibras

‘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