-Mannans of soybean meal:

Their enzymatic hydrolysis and the effect of

-mannanase on growth performance and immune
status of broiler chickens

A. Rogiewicz1, J. Naczmanski1, M. Alizadeh1, R. Patterson2,

and B. A. Slominski1
1University

of Manitoba, Winnipeg, Canada

2Canadian Bio-Systems, Calgary, Canada

2016 Poultry Science Association Annual Meeting, New Orleans, July 10-14

Outline
1. Introduction: the mannans and mannanase

2. In vitro mannanase evaluation

3. Growth performance and immune status of broiler

chickens

Introduction
Beta-mannans, found in common feed ingredients, are of high MW
molecules of mannose units with galactose side chains.
Palm kernel, copra meal, and guar meal contain 37, 26, and 9% of
mannans, respectively. SBM contains 1.6%, canola meal 0.5%, while
wheat or corn only 0.1%.
DDGS, as a co-product of brewers yeast (Saccharomyces cerevisiae)
fermentation, contain 1.3% of mannans. Yeast mannans mainly
consist of -1,6-linked mannose units, and are different from plant 1,4-mannans.

Introduction

Locust bean, guar gum, and SBM mannans are different due to
differences in galactose to mannose to ratio, i.e., guar gum - 1:1.7,
SBM - 1:1.8.
Due to differences in -galactomannan structures, and different
specificity of -mannananase preparations to such structures, different
degree of hydrolysis and the production of different hydrolysis products
would be expected.

- Mannanase activity as determined by

using different substrates (U/g1)

-Mannanase

Locust bean gum

Guar gum

Carob gum

54,618

1,782

17,042

27,576

2,076

14,143

17,922

1,776

33

138

21

59

30

12

1One

unit of activity is defined as the amount of enzyme which will produce one micromole of mannose reducing
sugar per minute under the defined assay conditions

NSP depolymerization of copra meal with two

different mannanase preparations (mg/g)

Enzyme
Control (no enzyme)

Arabinose Xylose

Mannose Galactose Glucose

Uronic
acids

Total
NSP

11.7

4.7

193.2

20.7

49.3

11.3

291.9

Mannanase A

5.8

3.9

75.7

12.0

17.3

10.0

114.5

Mannanase B

3.6

3.5

31.8

11.2

25.4

10.2

75.5

Introduction
Numerous studies on the elimination of the adverse effects of
poultry diets containing guar meal, copra meal, or palm kernel
meal by -mannanase supplementation have been published:

Ellis et al.,1995. Br. J. Nutr. 74: 539-556.

Kamran et al., 2002. Int. J. Poult. Sci. 1: 167-173.
Lee et al., 2003. Poult. Sci. 82: 1925-1931.
Daskiran, et al., 2004. Poult. Sci. 83: 662-668
Lee et al., 2005. Poult. Sci. 84: 1261-1267.
Sundu et al., 2006. Int. J. Poult. Sci. 5:1318.
Sundu et al., 2008. Int. J. Poult. Sci. 7: 511515.
Hussain et al., 2012. World's Poultry Science Journal, 68: 253-268.
Utami et al., 2013. Media Peternakan, 36: 192-196.
Ibuki et al., 2014. Anim. Sci. J. 85: 562-568.
Shastak et al., 2015. World's Poultry Science Journal, 71: 161-174.

Mannanase mechanisms of action

Mechanisms of action proposed for the positive responses
observed from dietary -mannanase supplementation:
1. Elimination of negative effects associated with viscous properties of guar
and copra meals.
2. Release of energy from -mannan hydrolysis products.
3. Elimination of the negative effect of -mannans on glucose absorption
and insulin production.
4. Suppression of the pathogenic bacteria proliferation by -mannan
hydrolysis products.
5. Prevention of energy taken away from lean tissue growth due to the
elimination of a -mannan-induced innate immune response.

Feed-Induced Immune Response (FIIR) theory

SMB -galactomannans trigger the metabolically costly stimulation
of the innate immunity called FIIR.
High MW mannan, acts as a Pathogen Associated Molecular
Pattern that activates the innate immune system. It can consume
around 3% of total metabolizable energy.
Immune stimulation affects the growth performance diverting the
metabolic energy away form growth.
The -mannanase is an energy-sparing enzyme and by reducing the
molecular weight of soluble -galactomannan decreases the immune
response thus helps spare some of the potentially wasted energy.

NSP content of SBM

%, as-is basis

16

14.8

%, as-is basis

12

1.1
0.1

0
Total NSP

Galactomannan

Water-soluble galactomannan

In vitro mannanase evaluation

SBM (100 mg) + Enzyme (1 mg)

Incubation at pH 5.2 and 45C for 5h

80% EtOH addition and centrifugation

EtOH-solubles

EtOH precipitate

discarded

NSP analysis

SBM NSP depolymerization using different

mannanase preparations
200

160

159 159
151

158

152

164 167 162

159
156

161 158
150

150

120

mg/g
80

40

0
Control

Multi-carbohydrase

Isolation of water-soluble -mannans of SBM

SBM (100 g) + water (500 g)

Slurries mixed overnight and centrifuged

Water-insolubles

Water-solubles

discarded

collected

Absolute EtOH added to bring to 80% and centrifuged

Supernatant decanted
discarded

Retente (EtOH-insoluble)
freeze-dried

Component sugar profile of water-soluble NSP

isolate from SBM

1. Rhamnose + Fucose
2. Arabinose
3. Xylose

5
1

2
4
3

2.0 %
15.3 %
2.2 %

4. Mannose

15.0 %

5. Galactose

33.9 %

6. Glucose
7. Inositol (internal standard)
8. Uronic acids (not shown)

8.3 %
23.3 %

In vitro mannanase evaluation

SBM isolate (100 mg) + Enzyme (1 mg)

Incubation at pH 5.2 and 45C for 5h

80% EtOH addition and centrifugation

EtOH-solubles

EtOH precipitate

discarded

NSP analysis

SBM water-soluble NSP depolymerization using

different mannanase preparations
100
90

81.8
80

78.6 78.9 79.6 80.2

82.7

80.3

74.5

74

70

80.6

63.3

59.4

60

mg/g

77.2

51.1
50
40
30
20
10
0
Control

Multi-carbohydrase

SBM water-soluble galactomannan depolymerization

using different mannanase preparations
25.0

20.0

19.1
15.5

15.0

13.1

mg/g

14.1

15.5 14.9 15.5

14.8
14.8

16.5

16.4
14.5

14.1 14.4

10.0

5.0

0.0
Control

Multi-carbohydrase

Broiler Chicken Study

Hypothesis: SBM -galactomannans may be involved in

triggering the innate immune response by the animal which would
be minimized by -mannanase supplementation.

Objective: To investigate the effect of different mannanases on

growth performance and immune status of broiler chickens.

Broiler Chicken Study

One day Ross/Ross broiler chickens
Dietary treatments:
1. PC: SBM/Corn- 3000 kcal/kg ME (starter) and 3100 kcal/kg ME (grower)
2. NC: SBM/Corn- 2900 kcal/kg ME (starter) and 3000 kcal/kg ME (grower)
3. NC + Multi-carbohydrase
4. NC + Mannanase B
5. NC + Mannanase K
6. NC + Mannanase M

Each diet was fed to 10 replicate pens of 5 birds each from 1 to

21 day of age

Broiler Chicken Study- Diets

Ingredient
Corn
SBM
Fishmeal
Oil
Minerals
L-Lysine
DL-Methionine
Threonine
Vitamin and Mineral Premix
Calculated composition
Crude Protein, %
ME, kcal/kg
Available Phosphorus, %
Calcium, %
Available Lysine, %
Available Methionine, %

Starter (1-10 days)

Grower (11-21 days)

PC
53.90
35.00
5.00
1.79
2.18
0.08
0.15
0.10
1.50

NC
56.10
34.60
5.00
0.00
2.18
0.08
0.15
0.09
1.50

PC
56.80
31.50
5.00
2.81
1.88
0.03
0.13
0.05
1.50

NC
59.00
31.21
5.00
0.91
1.88
0.03
0.12
0.05
1.50

22.99
3001
0.96
0.48
1.28
0.51

22.99
2900
0.96
0.48
1.28
0.51

21.50
3100
0.87
0.44
1.15
0.47

21.50
3000
0.87
0.44
1.15
0.47

Broiler Chicken Study

Immune status determination

Spleen and bursa of Fabricius relative organ weight;
Blood IgG, IgM and IgA level determination;
Duodenum, jejunum and ileum IgA level determination.

Growth performance of broiler chickens

1-21 d of age

Diet / treatment

Body Weight Gain

(g/bird)

Feed Intake
(g/bird)

FCR
(g feed/ g gain)

Positive Control

779.8

1024.3

1.32

Negative Control (NC)

774.8

1047.5

1.36

NC + Multi-carbohydrase

803.0

1037.0

1.30

NC + Mannanase B

806.2

1054.4

1.31

NC + Mannanase K

797.3

1061.8

1.33

NC + Mannanase M

764.9

1016.0

1.33

P- value

0.783

0.573

0.619

No statistically significant difference in growth performance was observed

Immunoglobulin content in serum and small

intestine of broiler chickens (g/ml)

Serum

Intestine

Diet / treatment
IgG

IgA

IgM

IgG

IgA

IgM

Positive Control

583

131

146

13.7

151

5.4

Negative Control (NC)

632

143

146

16.8

205

6.7

NC+ Multi-carbohydrase

548

125

129

14.6

168

6.1

NC + Mannanase B

636

157

124

15.2

196

6.4

NC + Mannanase K

556

140

143

14.2

161

6.1

NC + Mannanase M

471

125

127

12.8

127

5.8

0.063

0.129

0.402

0.770

0.570

0.600

P- value

No statistically significant differences were observed

Relative weight of immune organs (g/kg BW)

in 21 d old broiler chickens

Diet / treatment

Spleen

Bursa of Fabricius

Positive Control

50

0.84

2.62

Negative Control (NC)

48

0.81

2.41

NC + Multi-carbohydrase

49

0.79

2.56

NC + Mannanase B

48

0.76

2.30

NC + Mannanase K

48

0.71

2.47

NC + Mannanase M

50

0.80

2.27

0.153

0.096

P- value

No statistically significant differences were observed

Summary

There was no effect (P>0.05) of enzyme supplementation

on growth performance, the relative weight of immune
organs (spleen and bursa of Fabricius), and the level of
immunoglobulin IgA, IgG, and IgM in serum and intestine.

Future research

1. Identification of the main and side activities of mannanase preparations.

2. Validation of the -mannanase specific activity towards
SBM galactomannan.
3. Further validation of the energy-sparing effect due to
elimination of the mannan-induced innate immune
response.

Thank you