ADVANCES IN ENZYME SUPPLEMENTATION IN LIVESTOCK

Presented by

Nihar Ranjan Ghosh

Introduction
Demand for meat, milk and egg tumbled, so did feed production. Digestive process is nowhere near 100 percent efficient. Monogastrics do not produce the enzymes to digest fibre (Sheppy, 2001).

Young pig can not digest sucrose or more complex sugar.

Cont.
Increased digestibility of fibrous forages (Lewis et al., 1995; Yang et al., 1999) Increased ruminal microbial protein synthesis (Lewis et al., 1996) Improved growth rate, FCE, milk yield and milk composition (Chamberlain and Robertson, 1992; Beauchemin et al., 1995 )

Why enzymes in animal feeds

To break down anti-nutritional factors To increase availability of starch, protein and minerals To stimulate endogenous enzymes To break down specific chemical bonds that are not usually broken down by animals own enzymes

Feed enzymes in use today

-glucanase Xylanase Amylase Cellulases -galactosidase Phytase Proteases Lipases (Cowan, 1992; Ogdon, 1995)

ANTINUTRITIONAL EFFECTS OF NON STARCH POLLYSACCHARIDES Encapsulation of nutrients

Increase secretion of digestive juice

NSP
Excessive deconjugation of bile salt

Enlargement of digestive organs Nutrient digestion

Interact with the glycocalyx of intestinal brush border

Inhibition of protein breakdown

Producing a thickening of the rate limiting unstirred water layer

Results in nutrients absorption

Mode of action of cellulase and xylanase

Endoglucanase(CMCase) (Wood and Bhat, 1988) Cellobiohydrolase (Gilkes et al., 1997) -glucosidase (Christakopoulos et al., 1994) G-G; G-G-G-G; -G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G

Xylanase
(Coughlan and Hszlewood, 1993)

X-X-X-X-X-X-X-X-

-glucanase reduces viscosity

Glc1_ 4Glc1_ 4Glc1_ 3Glc1_ 4Glc1_ 3Glc1_ 4Glc1_ 4Glc
-glucanase

Oligosaccharides+ glucose

Anti-nutritional effect of phytate

OH O Starch Protein P O O Ca2+ OH O P O O H O Zn2+ H O Ca2+ O P O H OO - O C CH2 Protein

CH2
NH3+ O

H O O H

H O P O

O P O-

O O P OH CH2OH Starch O-

Bacteria Bacillus subtilis Lactobacillus acidophilus L.plantarum Streptococcus faecium Fungus Aspergillus oryzae A. niger Trichoderma reesei Saccharomyces cervisiae(Muirhead,1996) Transgenic plants e.g.xylanase from tobbaco plant (Herber et al., 1995; Liu et al.,1996)

Source of enzymes

Effect of -glucanase* on ileal and fecal digestibility in poultry fed hulless barley diet
Item Ileal digestibility Fecal digestibility

Control
Digestibility(%)

-glucanase

Control

-glucanase

DM
OM CP GE
-glucan

60.1
63.0 65.2 64.9

66.7
69.6 73.5 71.1

84.6
87.0 80.5 84.6

86.4
88.9 85.9 87.2

80.1
79.4 69.9

92.1
84.3 76.7

99.4
88.8 82.8

99.5
86.2 86.9

Arginine Valine

*2% -glucanase supplementation

(Li et al., 1996)

Effect of enzyme supplementation on BW gain, digesta viscosity, fat digestibility in broiler chicken feed hulless barley
Item HB HB+Enzyme * (0.025%)

Body weight gain(g) 0-2 weeks 6-8 weeks Feed conversion (F/G) 0-2 weeks 6-8 weeks Digesta viscosity(rel.) 2 weeks 8 weeks Fat digestibility(%) 2 weeks 8 weeks
53.2 69.4 76.3 74.1 2.59 1.74 1.49 1.42 1.95 1.8 1.54 1.79 142 905 284 870

* -glucanase+xylanase+ lipase

(Salih et al., 1991)

Effect of phytase on broiler chicken performance fed corn-soy diets

Item T1 (0.45% NPP) T2 (0.35% T3 (0.25% NPP) NPP) T4 (0.25% NPP+600U/kg crude phytase) T5* (0.25% NPP+600U/kg crude phytase)

Weight gain
(g/bird) 1-35d

1218.8

1089.5

736.8

1001.4

961.3

Feed intake
(g/bird) 1-35d

1967.1

1755.5

1286.1

1758.4

1680.0

Mortality%
1-35d

10

13.3

36.7

23.3

15.0

Feed/gain(g/g)

1.61

1.62

1.75

1.76

1.76

* Commercial preparation

(Paik et al., 1999)

Effect of xylanase on total population of bacteria in small intestine of 21 day broilers

12 10 8 6 4 2 0 Coliforms Enterococci Total Control Xylanase

(Vahjen et al., 1998)

Enzymes on digestibility of pig fed hulless barley based diets (Baidoo et al., 1998)
Item
Ileal digestibility(%) DM Energy CP NDF Total NSP Total tract digestibility(%) DM Energy CP NDF 76.7 75.0 66.2 49.8 80.3 79.4 72.0 54.2 56.0 57.1 57.6 17.6 6.4 64.5 63.3 61.8 35.4 33.8

Control

Enzyme*

* Cellulase (11080U/kg)+ -glucanase(27170U/kg) +xylanase(43350U/kg)

Effect of enzyme addition to a wheat based diet with a high extract viscosity, piglets 10-25kg
Wheat extract viscosity* Low(Positive control) High Daily gain(g) 388 354 Daily feed(g) intake 717 727 Feed: gain

1.85 1.92

High+ xylanase
(5000U/g)

462
480

766
794

1.66
1.65

High+ xylanase+ protease (500U/g)

*Low=1.3 mPas; high= 3.3 mPas.

(Dusel et al., 1997)

Phytase on digestibility of CP, AA, Ca and P in growing pig

Diet 1 2 3 4

Protein source
Phytase (Units/kg) Digestibility (%)

SBM
0

SBM
500

SBM
1000

SBM
1500

CP
Lysine

63.5
89.9

67.5
90.7

68.9
88.8

69.2
88.7

Ca
P

63.5
49.8

67.5
64.3

68.9
66.9

69.2
69.8

(Traylor et al., 2001)

Protease against ANFs

Prime target of proteases in soybean are the ANFs, such as residual trypsin inhibitor, lectins and antigenic protein. (Beal et al., 1999) 0.1% protease added to soybean meal significantly improve ADG, FCR, average daily feed intake in pig and poultry. (Rock et al., 1996; Beal et al., 1998)

Mode of action of exogenous enzymes in ruminants

Rumen Microbial numbers Microbial attachment Particle size Rate of passage Digestion

Small intestine Viscosity Digestion

Large intestine Digestion

(McAllister et al., 2001)

Effect of enzyme* supplementation on intake and nutrient digestibility

Item diet

control
Digestibility(%) DM OM ADF NDF 64.7 65.9 37.6 43.1

Low level enzymes(1.22L/T)

High level enzyme(3.67L/T)

67.3 68.1 42.5 44.2

64.7 66.0 40.3 39.9

Intake of digestible nutrients

DM
OM Predicted increase in milk yield (kg/d)

13.3
12.2 0.0

14.7
13.5 4.5

14.0
13.0 2.3

*Xylanase+-glucanase+cellulase+hemicellulase+protease

(BASF Corporation, 1998)

Effect of Enzyme supplementation on milk production of lactating cows

Item Control DMI (kg/d) 18.7 Milk production(kg/d) Actual 35.9 4% FCM 34.4 Milk composition % Fat 3.87 Protein 3.24 Lactose 4.73
* Cellulase+xylanase (1.3 g/kg TMR)

Enzyme* 19.0 39.5 35.8

3.37 3.03 4.62

(Rode et al., 1999)

Performance of lactating cows fed forages treated with enzyme

Enzyme treatment Item control EA2* DMI (kg/d) 22.0 22.5 Milk (kg/d) 37.0 39.5 3.5% 32.5 35.4 FCM(kg/d) Fat% 2.80 2.91 Protein% 3.14 3.19
* 3500U CMCase+16000U xylanase/kg forage DM ** 8800U CMCase+40000U xylanase/kg forage DM

EA5** 21.8 36.2 30.5

2.52 2.96 (Kung, 2000)

Enzymes* on dry matter intake, milk production and composition of lactating cows
Item DMI (kg/d) Diet control LH(1g HH(2g HT(1g E/kg conc.+ E/kg hay) E/kg hay) 1g E/kg hay) 20.4 20.7 20.7 20.8

Milk (kg/d)
4% FCM (kg/d) Fat% Protein% Lactose%

23.7
22.4 3.79 3.36 4.56

24.6
22.9 3.70 3.41 4.61

25.6
24.6 3.78 3.48 4.60

25.3
24.2 3.76 3.49 4.62

*31U Cellulase+43.4U xylanase/gm

(Yang et al., 1998)

Fibrolytic enzyme additive to alfalfa hay in feed lot cattle

Item 0
E1(1000U xylanase+ 40U cellulase/ kg)

ENZYME
E2(2000U xylanase+ 80U cellulase/kg) E3(3900 E4(7900 U U xylanase xylanase + 156U + 316U cellulase cellulase/ /kg) kg)

ADG(kg/d)

1.03

1.27

1.28

1.34

1.19

DMI(kg/d)
Feed efficiency
(kg DMI/kg gain)

10.2
9.9

10.8
9.0 35.5

10.5
8.7 37.4

11.7
8.5 31.8

10.9
9.6 33.0

ADF digestibility(%) 32.2

(Beauchemin et al., 1995)

Effect of exogenous enzymes on adhesion of probiotic bacteria

30 25 20 15 10 5 0 LGG LcS La1 Bb12 Control Treatment

L. rhamnosus GG (LGG) L. casei strain Shirota (LcS) L. johnsonii La1 (La1) Bifidobacterium lactis Bb12(Bb12) Pepsin+ parapepsin+ lysozyme
(Ouwehand et al., 2001)

Enzyme screening and selection strategies

Natural isolate screening

Molecular screening Process involve N-terminal sequencing, construction of probes and PCR (Dalbarge and Lange, 1998)

Engineering microorganisms for enzyme production

Classical mutagenesis
Mutation of DNA of organism by UV light or chemicals

Diverse population of cells obtain

Screening to identify cells having mutations conferring desirable traits

(Clarkson et al., 2001)

A. niger over expression vector for fae (deVaries et al., 1997)

A. niger glaA terminator

xba 1 faeA expression vector A. niger faeA

BgI II A. nidulans pyrG (marker)

A niger glaA promoter

Plasmid

Cleavage with endonuclease

DNA ligase
Recombinant vector Transformation Cell propagation

Chromosome

GENETIC ENGINEERING TO ALTER ENZYME EXPRESSION

Deletion of the T. reesei cellobiohydrolase 1 gene

Cbh1 gene upstream flanking sequence

Pyr4 gene

Cbh1 gene 3 downstream flanking sequence

chromosome

Cbh1 gene

Pyr4 gene

X
Pyr4 gene replaces in deleted cbh1 gene in the chromosome

Cbh1 gene

Pyr4 gene

Cbh1 gene is lost

Cbh1 gene

Industrial processing of exogenous enzyme

Stored culture

Seed flask

Transfer vessel

Seed fermentor

Production fermentor

Purification, decolorization & finishing

Collection of enzyme by micro filtration

Harvesting & cell separation

Areas requiring additional research

Satisfactory enzyme assay for monitoring quantities of enzyme present in complete diets. Determination of site and mode of action of specific enzyme in different species. Production of new enzymes. Generation of alternative source of enzymes.

Conclusion
Enzymes when properly used can produce significant improvement in poultry and pig performance and reduce the excretion of undigested nutrients. More comprehensive studies are needed to elucidate the mode of action of fibrolytic enzymes additive in ruminant diets, so that enzyme preparation can be formulated and applied to produce consistent and optimum responses.

Cont.
Concurrent development in biotechnology area will result in feed application for enzyme remaining an era of considerable interest.

Animal response versus the cost of enzyme should be considered to make the process feasible.

Milk production and composition of cows fed enzyme treated forages

Diet
Item Milk (kg/d) 3.5 % FCM (kg/d) Fat % Protein % Lactose % 1 25.1 25.4 3.70 3.28 4.92 2 26.2 27.3 3.83 3.37 4.92 3 26.0 28.0 3.92 3.43 4.94 4 27.8 29.7 3.84 3.36 4.93

Schingoethe, 1999

Effect of exogenous enzymes on VFAs

Item control Low level enzyme High level enzyme

VFA (mole/100 mole) Acetate 62.0 Propionate 23.4 Butyrate 11.0

64.8 20.1 11.4

62.5 24.1 9.7

(BASF corporation, 1998)

Effect of carbohydrase on pig nutrition

Referances
Officer(1995)

Basal diet
Wheat, fish meal, soybean Wheat, barley, maize, soybean

Enzyme(s)
Protease; lipase; amylase; glucanase; cellulase; pentosanase

Responses
Growth performance (C:299g/d; E:319g/d); Feed:gain (C:1.54; E:1.4) Growth performance (C: 575g/d; E: 581g/d) Feed:gain(C:1.67; E: 1.59) Growth performance (C:354g/d; E: 480g/d) Fecal digestibility(%) (C: 80.1; E: 83.1)

Partridge et al.(1998)

Xylanase; -glucanase; amylase; pectinase

Jeroch et al.(1999)

Wheat, rye, barley, soybean meal

Wheat,beet pulp, soybean meal

Xylanase; protease

Gill et al.(2000)

Xylanase; amylase; pectinase; -glucanase; pectinase

Feed:gain(C:1.53; E: 1.48) Fecal digestibility (%)of protein (C: 72.1; E: 73.9)

Enzyme
Enzymes are naturally occurring
biocatalysts produced by living cells

to bring about specific biochemical

reactions

Desirable characteristics of feed enzymes

High specific activities.

High level of resistance to heat, PH, and

proteolytic enzymes.

Low production cost.

Long self-life under ambient storage condition.

Lack of appropriate binding to feed matrix.

(Marquardt and Bedford, 2001)

Mode of action of phytase

H2PO4 H2PO4

H2PO4
H H2PO4 H H Phytase +H2O

Myo-inositol + myoinositol penta, tetra, tri, di and mono phosphate + inorganic phosphorous

H2PO4 H H2PO4

Method of application

Indirect method

Direct method

Influence of xylanases on performance and viscosity of poultry fed wheat diets

Intestinal viscosity FCR

6
Intestinal viscosity (cp)

5 4 3

1.61 1.51 1.41

2 1 0 Control Enzyme 1 Enzyme 2

1.31

(Cowan et al., 1996)