FOR INTERNAL USE ONLY

Definition of a protease
• Protease = Peptidase = Proteinase = Proteolytic enzyme

• Degrade protein by hydrolysis of peptide bonds

FOR INTERNAL USE ONLY

Occurrence and
functionality
• Analysis of complete genomes has shown that about
2% of proteins in all kinds of organisms are proteases

• Proteases have many different functions: processing of proteins,

protein turnover, cell division, metabolism, toxins…
A challenge for the broiler industry
• Demand for poultry meat is increasing

» population increase
» improved economic prosperity

• Protein is one of the most expensive raw materials in an

animal’s diet and is essential for growth

• Regional food security concerns

• Consumer and regulatory pressure to have a more

environmentally & animal friendly production is increasing

• Utilizing dietary protein sources more efficiently can help

produce more meat whilst meeting the environmental
challenges
Strategies to more efficiently utilize dietary protein

✓ Phase feeding
✓ Ideal protein ratios
✓ Low protein diets
✓ Synthetic amino acids
✓ Microbial (exogenous) protease enzymes
Proteases until now….

– Secondary activity in multi-enzyme

blends
• Difficult to separate protease
activity from de-caging effect
of carbohydrase
– Developed for other industries and
adapted for use in feed
• Poor gut stability
• Poor processing stability
• Poor product quality
– Inconsistent effects on animal
performance

5
Developing the ideal protease for feed

The ideal protease for feed

should: Gly Pro
His
Ala
• Remain stable in the gut
• Degrade many different
feed proteins
• Complement endogenous
proteases
• Remain stable during feed
manufacture
• Be compatible with other
enzymes

6
RONOZYME® ProAct – a unique
protease specifically developed for
feed

• Protease from Nocardiopsis

prasina produced in Bacillus
licheniformis
• Serine endo-peptidase
• >95% pure protease activity
• Broad specificity
• Stable at low pH/with pepsin
• Stable during feed processing
• Complements endogenous
enzymes such as pepsin and
pancreatic proteases
• Fully compatible with other
feed enzymes

7
FOR INTERNAL USE ONLY

Protease activity in commercial ‘protease

claiming’ products vary greatly
Protease activity determined using a highly sensitive assay
(Casein-FITC substrate, pH 8.3, 15 min, 37°C)

3x

7.5x

ELN-13-HALL-0016
FOR INTERNAL USE ONLY

Complementary and Synergistic with

endogenous proteases
In vitro digestion model –
a useful tool when screening for complimentary effects

Test enzyme

Analyse soluble

Feed protein

Analyse degree of
hydrolysis (DH)

The results reflect at the same time survival and action in the digestion model
 Complements endogenous enzymes

%, relative to the control

containing endogenous
(digestive) enzymes

Source: NOVOZYMES, Denmark

DH (Degree of protein hydrolysis) - this reflects the number of hydrolysed
peptide bonds and thereby the agressiveness of a protease
Soluble protein/total protein - reflects the ability of proteases to solubilise
proteins
15
FOR INTERNAL USE ONLY

Synergy with endogenous proteases

Protein solubilization
In vitro conditions: 120 * * *

Improvement relative to control (%)

115
- SBM-maize (30:70) 110
- 40°C 105

- pH 3/pepsin: 1hour 100

95
- pH 7/pancreatin: 4 hours 90

- Protease: 10 x rec. dosage 85

80

110 * Degree of hydrolysis

Improvement relative to control (%)

*
105
Products A, B, C and D represent
100
products claiming protease
95
activity as main activity or as side
90
activity
85

ELN-09-HALL-0008 & ELN-10-LNBR-0065 * Significant increase (P<0.05, all-pairwise Tukey-kramer HSD)

FOR INTERNAL USE ONLY

Compatibility with other feed enzymes

In vitro incubations with enzyme combinations

Phytate degradation by phytase and Xylan solubilisation by xylanase and

phytase + ProAct (20x) xylanase + ProAct (10x)

Corn-sbm diet: pH 6/30 min -> pH 3/pepsin/10 min Wheat bran: pH 7/3 hours
Phytase @ recom. dose, ProAct @ 20x rec. dose Xylanase @ recom. dose, ProAct @ 10x rec. dose

ELN-11-KPON-0001 & ELN-11-CAAO-0005

FOR INTERNAL USE ONLY

Protein digestion is not optimal

•pH of the gastro-intestinal tract

•Pancreatic enzyme development Bird age
•Solubility of protein
•Endogenous losses
•Antinutritional factors present in feed raw materials
– Lectins
– Trypsin inhibitors
•Antigenic proteins
•Refractory proteins e.g. Glycinin in soybean and Gliadin in wheat
•Passage rate
•Particle size
Exogenous protease can help improve AA
digestibility
• Work synergistically with endogenous proteases
(less specific than some endogenous enzymes)
• Improve the digestibility of amino acids in
ingredients
• Reduce endogenous losses
• Act on molecules refractory to endogenous
enzymes
• Improve gut health and integrity?
– Less undigested protein for bacterial
fermentation
– Degrade anti- nutritional factors
– Degrade antigenic proteins
– Increase gut tensile strength
 First approach
• Approach 1: Simple approach
– Increase digestible amino acid
levels of raw materials* from
3% to max 8%**
– Wheat based and low crude
protein diets - 3%
– Corn based diets - 4%

*Cereal and protein sources only

**Dependent on diet composition

23
Effect of supplementing RONOZYME® ProAct to
low CP/AA wheat soya based broiler diets
Species: Broiler
Country: Belgium
Results
Performance at 39 days
Objective
• To determine the effect on broiler
2400 1.65
performance of supplementing RONOZYME®

Weight g
ProAct to wheat soy based diets with

FCR g/g
2350 1.6
reduced crude protein and amino acid
content from 11-39 days of age. 2300 1.55

2250 1.5
Trial details T1 T2 T3
• Institute for Agriculture and Fisheries Treatment
Research (ILVO), Belgium
• Breed: 710 male Ross 308 Weight FCR
• Completely randomized design, 7
replicate pens (control diets) and 8
replicate pens (experimental diets) each
of 32 birds
• Diets: Wheat, corn, soyabean meal, full
fat soy based. Non heat treated mash. Conclusions & Benefits
• Treatments*: • The NC diet produced reduced performance at day 39
T1= Positive control (PC) diet • RONOZYME®ProAct supplementation to the NC diet gave superior
performance when compared to the PC diet
T2 = Negative control (NC) diet - 3% less
• RONOZYME®ProAct is an effective addition to wheat soya based diets
Crude protein/Amino acid than T1 from that also contain xylanase and phytase enzymes
11-39 days
T3 = T2 + 200 ppm RONOZYME® ProAct
* All diets contained xylanase and phytase

25 Source: ILVO July 2011

Effect of supplementing RONOZYME® ProAct to
low CP/AA wheat corn soya based broiler diets
Species: Broiler
Country: United Kingdom
Results
Performance at 42 days
Objective
• To determine the effect on broiler
performance of supplementing RONOZYME® 1.4
3.7 8
ProAct to wheat corn soy based diets with 6

Liveweight (kg)
reduced crude protein and amino acid 1.4

FCR (kg/kg)
3.7
8
content from 0-42 days of age. 2
3.6 1.4
8 7
3.6
1.4
Trial details 4
3.6 7
• Avian Science Research Centre, SAC, T1 T2 T3 1.4
6
Scotland Liveweight 1.4
• Breed: 1116 male Ross 308 FCR 6
• Completely randomized design, 12 1.4
replicate pens of 31 birds per treatment 5
• Diets: Wheat, corn, soyabean meal, full
fat soy based. Pelleted.
• Treatments*:
T1= Positive control (PC) diet Conclusions & Benefits
T2 = Negative control (NC) diet - 3% less • The NC diet produced reduced performance at day 42
Crude protein/Amino acid • RONOZYME®ProAct supplementation to the NC diet gave superior
performance when compared to the PC diet
T3 = T2 + 200 ppm RONOZYME® ProAct • RONOZYME®ProAct is an effective addition to wheat soya based diets
* All diets contained xylanase and phytase that also contain xylanase and phytase enzymes

Source26: SAC August 2011

 Back to basics…
The danger of ‘flat’ amino acid increases
• Disturbs the amino acid balance of
a well optimised diet (particular
problem in low protein diets)
• Performance is only improved by
the increase in the most limiting
amino acid (enzyme may not
deliver performance increase we
predict)
• Excess amino acids must be
deaminated and excreted (costs
energy, increases fat deposition,
bad for the environment)

An more accurate approach is needed

which considers amino acid balance
 Ileal digestibility – ‘room for
improvement’
• Digestibility meta-analysis included 804 data
points from 25 independent experiments

• Mean response to ProAct was 3.8% ranging from

5.6% for Thr to 2.7% for Glu

• AME was significantly increased by 49 Kcal/kg

and fat dig by 1%

• Inherent digestibility in the control diet

explained around 47% of the variance in response
= ‘room for improvement’

• When digestibility <70% then ProAct improves in

90% of cases by around 10%

• When digestibility >90% then ProAct improves in

60% of cases by around 2%

• Some ingredients do not follow the trend e.g.

rapeseed meal, sunflower

Page 30
Practical implementation into diet formulations

More precise/ initial implementation

more time consuming
1 Use of ingredient specific DIF values to
upgrade raw materials in the LCF

Use of matrix value calculation tool

2

3 Flat (e.g. 3/4%) matrix or DIF value per Rm

Less precise/implementation
quick

• Balance of precision and simplicity

• Balance of performance and cost saving
• User specific
Endogenous loss with and without a
mono-component exogenous protease
mg of AA/ kg DM Feed intake

Mateos et al, (2014)

• Proteases appear to assist the bird with recovery of endogenous protein or

even reduce endogenous secretion
Using high protein levels increases risk of
gut health problems

• Increase in protein also results in higher undigested protein

levels in the diet
• Protein impairs balance in gut microflora
– Increased protein levels increase the risk for imbalance microbial
community (Laudadio et al., 2012)
– Crude protein, protein source and amino acid content of diets affect
growth rate of C. perfringens (Drew et al., 2004)
– Protein in lower part GIT results in more proteolytic fermentation
– High protein increase risk for lower gut integrity
• Result: high protein results in increase of wet litter
(observation in practice)
FOR INTERNAL USE ONLY

Can proteases help overcome problems

with anti nutritional factors in SBM?
▪ Anti nutritional factors negatively impact animal performance

▪ Soybeans contain anti nutritional factors

▪ Kunitz trypsin inhibitor (KSTI) – a strong inhibitor of trypsin
▪ Bowman-Birk inhibitor (BBI) – inhibitor of trypsin & chymotrypsin
▪ Lectin (cause damage to intestinal enterocytes)

▪ KSTI, BBI and lectin are typically inactivated during toasting,

but levels are known to differ in commercial SBM