danisco animal nutrition - advances in...

Advances in Understanding Enzyme

Substrates in Feed and Available Solutions

Luis Romero, PhD

October 14th, 2015

Text starts with no bullet

Click once on

’Increase Indent’ for bullet

and click max. three times

for more bulletdesign

To get previous design back,

click on ’Decrease Indent’

Change Date and insert

Presentation Title under

>Insert >Header and Footer

and write in Fixed Date field

and field ‘Footer’ >Apply to All

Deactivate by clicking

off the checkboxes

Outline

Introduction. Mode of action and value of enzymes

Substrate and enzymes interactions in the gut

Phytase

Carbohydrases

Proteases

The microbiota as a key player

Capturing increased animal performance


Click once on












off the checkboxes

Reduced

performance +

mortality

There is a gap between genetic potential and commercial

performance in animal production

O

Production

frontier

Meat

(kg/flo

ck)

Feed, labor, other inputs ($)

Sub-optimal

production

Sub-optimal diet digestibility

Feed ingredient variation

Sub-clinical disease

Clinical disease

Environmental stress

Commercial

performance

Value

Potential

Romero, 2010


Click once on












off the checkboxes

Mode of action of exogenous enzymes in animals is not

limited to hydrolysis of one substrate

10/29/2015

Enzyme + substrate

• Digesta

• Physical structure

• Solubility of nutrients

• Host

• Feedback mechanisms

• Endogenous enzymes

• Gut development

• Immune response

• Microbiome profile and function

Balance and retention

of nutrients


Click once on












off the checkboxes

Screening

Small scale testing

Commercial application

Customer need

Assay and product

specifications

Understanding exogenous enzymes for animal nutrition

Customer

value

Disrupting

technologies Mode of action Systems biology


Click once on












off the checkboxes

Substrate and enzymes

interactions in the gut

10/29/2015


Click once on












off the checkboxes

Characterization of phytase activity in-vitro

Aspergillus niger E. coli

Citrobacter Braakii Buttiauxella sp.

Menezes-Blackburn et al. 2015. J. Agr. Food Chem.


Click once on












off the checkboxes

Maximum IP degradation in the digestive tract is now

reaching levels that are close to 90%, but multiple factors

affect maximum disappearance

10/29/2015

* 0, 500 or 1000 FTU/kg of Buttiauxella sp. phytase were supplied to broilers from 11 to 13 d of age

Li, Angel, et al. Submitted for publication, 2015

Factor Ca nPP PP Phytase Ca×nPP Ca×PP Ca×

Phytase nPP×PP nPP×

Phytase PP×

Phytase Ca×nPP×

PP Ca×nPP×

Phytase Ca×PP×

Phytase nPP×PP×

Phytase

Ca×nPP×

PP×Phytas

e

F Prob 0.0003 0.86 <0.0001 <0.0001 0.66 0.22 0.38 0.17 <0.0001 0.0024 0.12 0.27 0.88 0.15 0.79


Click once on












off the checkboxes

1420

970

295

44 9615 28

107

213

65

176 96138

280

200

400

600

800

1000

1200

1400

1600

1800

Starch Protein Fat Arabinoxylans Pectins Cellulose Resistant oligo-saccharides

Dig

est

ible

en

erg

y in

a c

orn

-so

y-D

DG

S d

iet

in b

roile

r ch

icke

ns

and

yo

un

g p

igs

(kca

l/kg

)

Digestible energy Non digestible energy

Undigested energy and protein substrates

10/29/2015

Non digestible energy ~ 820 kcal/kg

Realistic improvement ~ 440

Current improvement ~ 100 kcal/kg

Non digestible protein ~ 40 g/kg

Realistic improvement ~ 20 g/kg

Current improvement ~ 4 g/kg


Click once on












off the checkboxes

Mechanisms of action of carbohydrases and

proteases in broiler diets

Xylanase, beta-glucanase Reduced viscosity (Choct, 1999)

Improved access to cell contents (Cowieson,

2005)

Prebiotic effects (Fernandez et al., 2000)

Possible reduction of endogenous inputs (Satchithanandam et al., 1990)

Feed intake

Digestion

Feces

Endogenous inputs

Fermentation

Absorption

Production

a.a., starch, fat

a.a., NE

A X

X A

P

Protease Hydrolysis of dietary protein and increased protein solubility (Caine et al., 1998) Disruption of protein-starch interactions in corn (Mc Allister et al., 1993; Belles et al., 2000 )

Disruption of protein-fiber interactions (Pedersen, 2015)

Amylase Down regulation of pancreatic amylase (Jiang et al., 2008)

Augmentation of pancreatic amylase activity in young animals (Gracia et al., 2003) Improvement of digestion of resistant starch in corn and corn by products (Sharma et al., 2010) P

X SCFA


Click once on












off the checkboxes

Xylanases and beta-glucanases. Shifting the target from the

soluble to the insoluble fractions

Arabinoxylan and beta-glucan in some feed ingredients (% dry matter)

Source: Choct (2006); Danisco Non Starch Polysaccharide (NSP) database (2012)

2

7

12

17

22

27

Corn Wheat Barley Rye Wheatbran

WheatDDGS

CornDDGS

Soybeanmeal

Rapeseedmeal

Sunflowercake

Insoluble arabinoxylan Soluble arabinoxylan Total beta-glucan


Click once on












off the checkboxes

It is not just about solubilisation of fibre; it is about net energy

for the animal

10/29/2015

Enzymes: Solubilize fiber and release nutrients

Amino acids

& peptides Starch Fats

More nutrients absorbed Microbial fermentation

Availability of energy from fiber via SCFA production:

• Butyrate (2185 kj/mol)

• Propionate(1528 kj/mol)

• Acetate(874kj/mol)

Hemi-

cellulose


Click once on












off the checkboxes

There is considerable potential to increase protein

digestibility in commercial diets

10/29/2015

Ingredient * Crude

protein

ME (Kcal/kg) Main proteins Key amino

acids

% App.

dig**

Poultry Swine

Corn 8 0.82 3390 3350 Zein Leu, asp, glu

Wheat 11 0.81 3210 3415 Glutenin Glu

Soybean meal 48 0.85 2458 3140 Glycinin, beta-

conglycinin Phe, tyr, leu

Corn DDGS 27 0.65-

0.85 2800 3300

Globulin, glutelin, zein

Leu, asp, glu

Sorghum 11 0.68 3310 3230 Kafirin Pro, glu

Canola meal 38 0.77 2110 2600 Cruciferin,

napin Glu, asp

Sunflower meal 41 0.83 2310 2740 Helianthinin Glu, asp

Feather meal 85 0.50-

0.75 2880 2270 Keratin Ser, pro, gly

Meat and bone meal 50 0.65-

0.80 2530 2435 Collagen Gly, ala, pro


Click once on












off the checkboxes

Exogenous proteases

Serine proteases (3.4.21.xx)

Trypsin subfamily

proteases (3.4.21.4)

Subtilisin proteases

(3.4.21.62)

• Catalytic mechanism = His-Ser-Asp

• Alkaline pH optimum

• Higher specificity to

negatively charged

substrates

• Higher specificity to

hydrophobic substrates Aspartic acid

proteases

Metallo-protease

Cysteine

proteases

Most commercially available proteases fall into

this category


Click once on












off the checkboxes

Proteomics - a new tool

Proteomics is the large-scale experimental analysis of proteins present in a

biological sample. It usually relies on the extraction of the proteins from the

sample, their separation, protein digestion, followed by mass spectrometry

analysis

Proteomics has been applied to the study of food or feed protein digestion,

and the terminology “protein digestomics” has been put forward by Picariello

et al, 2013

• Le Gall et al. (2005)

• Identified two proteins in peas, lectin and albumin PA1b, that were

totally resistant to gastric and small intestinal digestion in pigs

• Fisher et al. (2007)

• Identified aggregated peptides of partly degraded β-conglycinin alpha

subunits in the undigested ileal residue of pigs fed soybean meal

10/29/2015

Improvements on amino acid digestibility due to enzymes are

proportional to ileal undigested amino acids in control diets

10/29/2015

y = 0.03x

R2 = 0.56

y = 0.03x

R2 = 0.42

y = 0.12x

R2 = 0.96

y = 0.13x

R2 = 0.94

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Apparently undigested fraction of amino acid (g/kg)

Ap

pare

nt

up

lift

on

am

ino

acid

dig

esti

bilit

y (

g/k

g)

XA-Corn/SBM XAP-Corn/SBM

XA-Corn/SBM/DDGS XAP-Corn/SBM/DDGS

met met

lys

lys

gln

gln

thr

thr leu

leu

asn

asn

cys cys

val

val

Romero et al., 2013


Click once on












off the checkboxes

Protein contributed a significant amount of energy in

response to enzymes, particularly protease, in 21-d broilers

IDE = ileal digestible energy

CS=Corn/Soy; WS=Wheat/Soy

XA = xylanase and amylase; XAP = XA plus protease

Mixed diets contained 10% corn DDGS and 5% canola meal

15 13 15 23 28 35 35 30 2 10 0

12

28 25

40 38

5

29 25

39

36

56 34 54

0

89

54

75

59

133

94

120

0

20

40

60

80

100

120

140

CS + XA CS + XAP CS Mixed+ XA

CS Mixed+ XAP

WS + XA WS + XAP WS Mixed+ XA

WS Mixed+ XAP

Imp

rovem

en

t o

f ileal d

igesti

ble

en

erg

y 2

1 d

(kcal/kg

DM

)

Starch Fat Protein IDE

Romero et al., 2015


Click once on












off the checkboxes

At 42 days, starch contribution was relatively greater, and

carbohydrase had smaller effects on protein contributions

23 30 38 34 38 50

67 78 4

20 8 18 11

20

24

34

25 30 21

40 18

42

0

20

40

60

80

100

120

140

160

180


CS Mixed+ XAP


WS Mixed+ XAP

Imp

rovem

en

t o

f ileal d

igesti

ble

en

erg

y (

kcal/

kg

DM

)

Starch Fat Protein

Romero et al., 2015






Click once on












off the checkboxes

Contributions of starch, fat and protein did not explain ileal

digestible energy in wheat based diets at day 42 d

23 30 38 34 38 50 67 78 4

20 8 18 11 20

24 34

0

25 1

30 21

40 18

42

35

90

32

96

134 147 149

275

0

50

100

150

200

250

300


CS Mixed+ XAP


WS Mixed+ XAP

Imp

rovem

en

t o

f ileal d

igesti

ble

en

erg

y (

kca

l/kg

DM

)

Starch Fat Protein IDE

Romero et al., 2015






Click once on












off the checkboxes

Proteases can have significant effects on total tract NSP

disappearance in broiler chickens

* A subtilisin protease at two doses (P5000 and P10000) and a combination of xylanase,

amylase and protease at three doses (XAP2500, XAP5000, XAP10000) were

supplemented in a corn-based diet from 14 to 21 d to broiler chickens

Olukosi et al, 2015


Click once on












off the checkboxes

Some practical implications

Net effects of enzymes on performance are greater in diets

with greater undigested substrates, which normally

correspond to diets with more fibrous ingredients.

Effects of exogenous enzymes on the digestibility of different

energy substrates overlap. Matrices are not additive.

Therefore, it is always better to work with enzymes

combinations with enough knowledge of mode of action and

robust matrices.

10/29/2015


Click once on












off the checkboxes

The microbiota as a key

player

10/29/2015


Click once on












off the checkboxes

24

You are bacterial and so are your birds and pigs!

Avian gut microbiota

Total bacterial pop. ~1014 cells (10x more than host cells”)

The avian gastrointestinal tract consists of many small ecological niches; either

mucosal or luminal

Gut bacteria are in the mucus layer at the epithelium and on digesta forming biofilms

Essentially sterile at hatch, the gut flora of an adult bird is relatively stable and difficult

to change by feed additives

Microbiota influences:

• Development and function of immune system

• Metabolism, appetite

• Disease

• Behavior


Click once on












off the checkboxes

Critical interactions of exogenous enzymes and the

gut microbiome

Direct effect

Critical interaction

Feed

enzymes

Increased

growth and

feed

efficiency

More

nutrients

digested

More

nutrients

absorbed and

metabolized Reduced

endogenous

inputs for

digestion

Reduced

populations

of pathogenic

bacteria

Prebiotic

effects

Reduced

endogenous

inputs for

immunity

Less

nutrients

available for

pathogens

Improved gut

integrity

Increased

fibre

digestion


Click once on












off the checkboxes

Enzymes might affect gut health through changes in

the available substrate and direct effect in the mucosa

• Xylanases have been shown to have pre-biotic effects in poultry

(Fernandez, 2000) and other species through selective stimulation of

beneficial bacteria and production of short-chain fatty acids

(SCFA) (Broekaert et al., 2011)

• Increased undigested protein appears to be a predisposing factor

for dysbacteriosis related to necrotic enteritis (Dahiya et al., 2007)

• Protease has been shown to improve performance of chickens

challenged with Eimeria spp. (Peek et al, 2009)


Click once on












off the checkboxes

Enteric disease is a limiting factor to the efficacy of

exogenous enzymes due to mal-absoption

281 311 308 304 315

38 57 94 122 130

305 191 110 49 -0.9

0

100

200

300

400

500

600

700

800

0 0.5 1 1.5 2

En

erg

y a

llo

cati

on

(k

cal/b

ird

/day)

Lesion scores (0-4)

Maintenance cost Added energy lost in excreta

Retained energy MEn intake

Teeter et al. 2011; Broussard et al., 2008

Energy partitioning of 42-48 d old broilers challenged with oocysts of three Eimeria species


Click once on












off the checkboxes

Combinations of enzymes and DFMs increase the

consistency of response in diverse levels of challenge

10/29/2015 Dersjant-Li et al.,

2014


Click once on












off the checkboxes

The development of deep sequencing techniques offers

completely new insights into the role of the gut microbiome

10/29/2015

Fraher et al., 2012

C Huttenhower et al. Nature 486, 207-214 (2012) doi:10.1038/nature11234

Carriage of microbial taxa varies while metabolic pathways remain stable within a

healthy population


Click once on












off the checkboxes

Capturing increased

animal performance

10/29/2015


Click once on












off the checkboxes

Capturing value in commercial conditions

• Selection of enzymes should be based on the effects on undigestible substrates

of base diets

• Enzymes combinations with significant, measurable and reliable activity levels

are preferable:

• Wide and consistent range of functionalities

• Higher chance of reliable net benefits in variable commercial conditions

• Proactive management of nutrient interactions are necessary:

• Effect of Ca on phytase activity

• Estimation of AME and protein quality

• Respond to seasonal or supply driven changes in ingredient quality

• Optimization of gut health in critical

10/29/2015


Click once on












off the checkboxes

Exogenous enzymes - Future R&D directions

Increase research on application knowledge

• Ingredient, animal and additive interactions

• Improvements on in-vitro simulations

Application of omics tools

• Proteomics. Increased digestion of undigested proteins

• Metagenomics. Increased energy from fibre; optimization of gut health

• Metabolomics and transcriptomics. Intestinal and systemic mechanisms

New applications of enzymes beyond digestion of nutrients

10/29/2015


Click once on












off the checkboxes

Thank you

10/29/2015 [email protected]

Copyright © 2012 DuPont or its affiliates. All rights reserved. The DuPont Oval Logo, DuPont™ Danisco® and all products denoted with ™ or ® are

registered trademarks or trademarks of E. I. du Pont de Nemours and Company or its affiliates.

Copyright © 2011 DuPont or its affiliates. All rights reserved. The DuPont Oval Logo, DuPont™, The miracles of science™ and all products denoted with ™ or ® are registered trademarks or trademarks of E. I. du Pont de Nemours and Company or its affiliates.

Images reproduced by E. I. du Pont de Nemours and Company under license from the National Geographic Society.

10/29/2015

danisco animal nutrition - advances in...

Documents