CHAPTER II

REVIEW OF LITERATURE
2.1 Corn/maize (Zea mays)
Cereal grains such as maize, wheat and barley are the main source of dietary
energy in poultry nutrition and maize is the most important grain in this respect. It
contains the highest amount of energy (ME 3350 Kcal/kg) among cereal grains and
is highly palatable. White, red and yellow are the three types of maize and among
all, yellow variety is commonly used for animal feed. Yellow maize unlike white
maize, provides carotene and xanthophyll pigment for coloration of egg yolk,
poultry fat and skin. Maize is also an excellent source of linolenic acid. The seed is
high in starch (65-70%), but low in protein (8.8%), fibre and minerals. Maize
protein is mainly deficient in tryptophan and lysine.
The energy value of corn is contributed by the starchy endosperm (65-70%),
which is comprised mainly of amylopectin and the germ, which contains most of the
oil. Most corn contains three to four per cent oil. The protein in corn is mainly as
prolamin (Zein) and as such amino acid profile is not ideal for poultry. Maize is also
more susceptible for infestation with mycotoxins producing fungi than other grains
such as wheat, sorghum and millets (Devegowda et al., 1998, 2004). The nutrient
composition of the maize is given in table 1. In India, the maize production is 11.5
million tones with the productivity of 1.7 tones / hectare.
2.1.1. Antinutritional effects
Maize can be included in the poultry diet up to 70 per cent. It has been
reported that at high levels, when fed for prolonged periods yellow carcass fat may
results due to the xanthophyll content. The non-starch polysaccharide composition
of the maize is discussed in the subsequent chapter.
2.2. Soybean meal (Glycine max)
Soybean meal (SBM) is an excellent source of protein and contains well-balanced
amino acid profile. This makes SBM a valuable ingredient in the diets of poultry. Protein
and energy content vary in soybean meal depending on protein level of the beans,
residual fat after processing and whether or not hulls have been removed. The protein

16
content of dehulled material ranges from 47.5 to 49.1 or more and material with hulls
ranges from 40 to 50 per cent with 44 per cent considered the norm.
SBM is an excellent source of lysine, tryptophan and threonine but is deficient in
methionine. The amino acid in corn protein and soy protein combine well to provide a
balanced mixture for most poultry requiring only minimal levels of synthetic methionine
to be used. Digestibility of lysine and methionine is over 89 per cent in properly
processed SBM. The energy level of SBM depends on residual oil, fibre content and ash
levels. ME levels for poultry have been estimated to be 120 to 250 Kcal/kg higher for
dehulled meal versus meal containing hulls (Rhonen Poulnec, 1993).
Properly processed SBM is an excellent ingredient that can be used as the sole
protein supplement for virtually any class of animal with no restrictions except perhaps in
piglet prestarter feed (20-25% max) or shrimp feed (15-20%). SBM is sometimes
adulterated with moisture (water) and rice bran, deoiled rice bran, urea and under
processed or other processed SBM. The nutrient composition is given in Table 1. The
production of soybean in World and India is given in table 2 and 3, respectively.
2.2.1. Antinutritional factors
Like most other high protein plant materials, SBM too contains
antinutritional factors (ANFs) like trypsin inhibitors (protease inhibitor),
hemagglutinins (lectins), phytate phosphorus and indigestible carbohydrates such as
oligosaccharides and nonstarch polysaccharides (Pack and Bedford, 1997;
Marsman et al., 1997; Zenella et al., 1999; Malathi and Devegowda, 2001; Graham
et al., 2002).
2.2.1.1. Trypsin inhibitors (Protease inhibitor)
The most problematic natural toxin of soybeans for poultry is trypsin inhibitor.
They will disrupt the protein digestion by rendering unavailable the digestible enzymes
trypsin and chymotrypsin and their presence is characterized by compensating
hypertrophy of the pancreas (Monari, 1998). At least, five trypsin inhibitors have been
identified, however, the principal TIs present in raw soybean are the Kunitz factor and
Bowman Brik factor, the latter is more resistant to the action of heat, alkali and acid.
Their average levels in raw soybean are of the order of 1.4 and 0.6 per cent, respectively.

17
1.Table Nutrient composition of corn and soybean meal

Soybean
Corn
meal
Major nutrients1
Crude protein (%) 8.3 48.0
ME (Kcal/kg) 3300 2400
Fat (%) 3.8 0.8
Fibre (%) 3.0 6.0
Calcium (%) 0.02 0.29
Total phosphorus (%) 0.28 0.65
Average phosphorus (%) 0.13 0.27
Linolenic acid (%) 1.9 -

Amino acid composition (%) 1

Lysine 0.24 2.80
Methionine 0.18 0.69
Methionine + cystine 0.35 1.39
Tryptophan 0.06 0.62
Arginine 0.39 3.28
Threonine 0.30 1.81

Mineral composition2,3
Chloride (%) 0.04 0.05
Mg (%) 0.15 0.26
Sodium (%) 0.05 0.01
Potassium (%) 0.38 2.02
Fe (%) 0.01 119 (mg/kg)
Mn (mg/kg) 9.00 29
Zn (mg/kg) 29.00 40
Se (mg/kg) 0.04 0.1
Vitamin composition2,3
Vitamin B12 (mg/kg) - 2.0
Vitamin. A (IU/kg) 2.40 -
Xanthophyll (mg/kg) 20.0 -
Riboflavin (mg/kg) 1.3 2.9
Vitamin E (mg/kg) 19.0 4.5
Choline (mg/kg) 60.0 2762
Biotin (mg/kg) 0.07 0.3
Thiamin (mg/kg) 4.4 4.5

18
1
Devegowda, 2005; 2Reddy and Bhosle, 2001; 3Leeson and summers, 2001

Table 2. World soybean production (2004-2005)

Country Million (%)

metric tonnes
United States 65.8 34
Brazil 53.5 28
Argentina 34.0 18
China 16.2 9
India 6.8 4
Paraguay 4.0 2
Other 9.8 5

Total 190.0 100

(American Soybean Association, 2004 – 2005)

Table 3. Soybean production in India (Million metric tones)

State 2003-04 2004-05

Madhya Pradesh 3.45 4.20
Maharashtra 1.7 1.87
Uttar Pradesh 0.02 0.02
Rajasthan 0.42 0.60
Karnataka 0.1 0.04
Chattisgad 0.04 0.04
Andhra Pradesh & others 0.14 0.08

Total 5.85 6.85

19
 (Solvent Extractors Asso. of India, 2004 – 2005)
2.2.1.2. Haemagglutinins / lectins
These are proteinaceous compounds present in soybean at a rate of one to three
per cent. Being the glycoproteins they have the ability to bind to cellular surface via
specific oligosaccharides and glycopeptides (Olivera et al., 1989) and have a relatively
high binding affinity to small intestinal epithelium (Pusztai, 1991), which results in a
reduction in nutrient absorption. Lectins can produce impairment of brush border
continuity and ulceration of villi, which may result in increased nitrogen losses and
depressed growth rate in young animals (Pusztai et al., 1990). Thus, the growth
depressant effect of lectins is believed to be primarily due to their damaging impact on
intestinal enterocytes and through appetite depression. However, these two
antinutritional factors are heat labile and are removed by heat processing.
ANFs in other plant protein sources – notably gossypol in cottonseed meal, both
glucosinates and erucic acid in rapeseed meal, alkaloids in lupin and tannin in peas are
not heat labile and must be managed in other ways. The processing of SBM involves a
series of heat treatment that include conditioning, rolling, flaking and extraction followed
by desolventizing and roasting. Roasting is the process in which SBM is cooked at 105
to 1100C for a period of 15 to 30 min and is a critical step in controlling the nutritive
quality of SBM.
Undercooking results in insufficient destruction of TI and other ANF, whereas
overcooking decreases amino acids availability, lysine in particular, is very heat sensitive
and subsequently leads to a reduced growth performance (Araba and Dale, 1990; Parson
et al., 1991). The industry monitors SBM quality by using urease activity to detect
underheating and potassium hydride (KOH) solubility to detect over heats. Destruction
of the urease enzyme activity is correlated to distruction of TI and other ANFs. The level
of urease activity should not exceed a pH rise of 0.2 units (Devegowda, 2005; Monari,
1998). To measure KOH solubility, soy products are mixed with 0.2 per cent KOH and
the amount of nitrogen solubilised decreases as heating time increases, indicating
decrease amino acid availability. The solubility index should be 80 to 85 per cent in
commercial SBM (Araba and Dale, 1990).

20
2.2.1.3. Nonstarch polysaccharides (NSPs)
Polysaccharides consist of macromolecular polymers of manosaccharides joined
by a specific type of linkage called glycosidic bond. NSPs are defined as polymeric
carbohydrates, which differ in composition and structure from amylose and amylopectin
(Gruppen, 1996). In starch, glucose molecules are mainly joined by (1-4) bonds with a
smaller number of (1-6) bonds. NSPs contain glycosidic bonds other than (1-4) and
(1-6) bonds present in starch. The nature of the bonds determines their susceptibility to
cleavage by avian digestive enzymes. Glycosidic bonds other than (1-4), (1-6), (1-2)
and (1-1) are resistant to digestive enzymes, but can be cleaved by microbially derived
enzymes (Smits and Annison, 1996). These NSPs have high molecular weight ranging
from 8000 to a million.
The non-starch polysaccharides content of corn and soybean meal is given in
Tables 4-8.
Table 4. Free sugars and neutral non-starch polysaccharides (NNSP) composition of
SBM (g/kg DM)
Soybean meal
Sugars Free sugars Insoluble Soluble Total
NNSP NNSP

Rhamnose 0.65 1.76 0.33 3.86

Fucose 0.50 2.21 0.14 4.60

Ribose 0.11 0.34 0.70 1.39

Arabinose 0.79 25.09 1.57 51.75

Xylose 0.51 11.64 0.39 23.67

Mannose 4.43 4.83 1.40 11.07

Galactose 18.43 42.00 4.98 88.98

Glucose 34.90 25.10 2.13 52.33

Total 59.53 100.95 10.45 170.93

(Kocher et al., 2002)

21
 Table 5. Total pentoson, cellulose, pectin and total NSP content of different feed
ingredients
Total pentosan Cellulose Pectin Total NSP
Ingredients (%) (%) (%) (%)
Maize 5.35 3.12 1.00 9.32
Sorghum 2.77 4.21 1.66 9.75
Finger millet 3.31 3.03 1.76 9.40
De oiled ricebran 10.65 15.20 7.25 59.97
Soybean meal 4.21 5.75 6.16 29.02
Peanut meal 6.11 6.55 11.60 29.50
Sunflower meal 11.01 22.67 4.92 41.34
Rapeseed meal 8.85 14.21 8.86 39.79

(Malathi and Devegowda, 2001)

Table 6. NSP composition of corn and soybean meal

LMW sugars Maize Soybean meal

22
 Manosaccharides 4 7
Sucrose 13 70
Rabinose 231 10
Xtachyose 20 47
Total sugars 690 3
Starch 6 137
Fructose 1 27
NSPb - -
-glucose 9 63
S-NCPc 0 1
Rhamnose 3 9
Arabinose 2 2
Xylose 2 5
Mannose 1 16
Galactose 1 6
Glucose 1 25
Uronic acids 66 92
I-NCPd 0 2
Rhamose 19 17
Arabinose 28 17
Xylose 1 8
Mannose 4 25
Galactose 9 1
Uronic acids 6 23
Cellulose 22 62
Total NSP 97 217
Dietary fibre 11 16
CHOe and lignin 108 233
Analysed 823 400
Calculated 830 416

(Khudsen, 1997)

a
Low molecular weight sugar; b Non-starch polisaccharide
c d
soluble non-cellulosic polysaccharides; insoluble non-cellulosic
polysaccharides; e carbohydrate

23
Table 7. Non- starch polysaccharide (%) content of some feed ingredient
Ingredient Soluble NSP Insoluble NSP

Maize 5.5 3.8

Jowar 4.3 5.5

Ragi 6.7 2.7

Groundnut extrn. 8.6 20.9

Soybean meal 13.90 16.40

Sunflower meal 4.60 23.10

Rapeseed meal 11.30 34.80

(Sreedhara, 2000)

24
Table 8. Soluble, insoluble and total NSPs content of different feed ingredients (% DM)
(1Ramesh and Devegowda, 2004; 1Kumar, 2002; 2Devegowda, 2005)

Pectin Cellulose Oligosacch2 Total2 Soluble2

Ingredient Pentosan (%)1
(%)1 ( %)1 arides (%) NSP (%) NSP (%)
Maize 5.12 0.80 2.91 0.3 9.3 5.5

Jowar 2.87 1.52 3.82 0.1 9.8 4.3

Rice broken 1.18 0.53 0.73 - 5.2 -

Ricebran, deoiled 10.23 5.62 14.40 - 59.9 -

Soybean meal 4.20 6.50 5.90 5.1 2.90 9.1

Groundnut extraction 7.11 11.00 6.20 3.1 29.5 8.6

Sunflower extraction 10.10 5.50 23.50 2.9 41.3 4.4

Rapeseed extraction 7.30 10.92 11.50 0.4 39.8 5.4

Double zero rapeseed 3.05 9.41 9.20 - - -

meal

25
2.2.1.3.1. Structure of plant cell wall
The starchy endosperm of any grain is covered serially of subaleurone layer, aleurone
layer and pericarp of plant cell wall (Fig 1) in a highly ordered way and consists of
different NSPs, which are closely associated with other polysaccharides and
noncarbohydrate material such as protein, lipids and lignin (Smith and Annison, 1996).
2.2.1.3.2. Classification of NSPs
The NSPs contains arabinose, xylose, mannose, glucose and galactose residues as
the principle constituent sugars.
1. Cellulose: Cellulose is the most abundant organic compound in nature, comprising
over 50 per cent of all the carbon in vegetation. Cellulose is a linear unbranched chain of
(1-4) linked D-glucose molecules. It is of high molecular weight and cellulose of up to
7,000 to 10,000 glucose units can be found in plant materials.
2. Hemicellulose: Hemicellulose is the second most abundant plant structural
polysaccharides. They are found most often as heteropolymers and less commonly as
homopolymers of manosaccharides and mainly include D-xylose, D-mannose, D-
galactose, L-arabinose, D-glucourinic acid, and D-glucose, etc.
The commonly occurring hemicellulose includes,
a. Arabinoxylan (Pentosan): Main chain is made of (1-4) xylose units and side chain
made of (1-3) linked arabinose units attached to C-3 position of the xylan main
chain.
b. -glucans: Consists of linear chain of glucose units joined by both (1-3) and (1-
4) linkages.
c. Xyloglucans: Made of (1-4) linked D-glucans main chain to which side chains of
D-xylose units are attached at C-6 positions.
d. Galactomannan: Made of (1-4) linked D-mannans backbone to which D-
galactose side chains are attached at C-6 position.
3. Pectic substances
a. Polygalactouranans: Main chain is made of (1-4) linked D-galactouronic acids
and side chains consists of either (1-3) linked D-xylose, (1-6) linked D-galactose
or (1-4) linked L-arabinose.

26
 b. Rhamnogalactouronans: Similar to polygalactouranans except that the main chain
in addition to D-galactouronic acid contains (1-2) linked rhamnose residues
resulting in a bent macromolecule.
4. Oligosaccharides (-galactosides): Successive addition of -D galactosyl residues to
sucrose primer lead to formation of raffinose, stachyose, verbacose and ajugose.

2.2.1.3.3. Antinutritive effects of NSPs in poultry

1. Increases intestinal Viscosity
Ingestion of soluble NSPs like arabinoxylans, -glucans and pectins etc.,
increases the digesta viscosity in broilers. The soluble NSPs increase the viscosity by
directly interacting with water molecules. At higher concentrations, the molecules of the
NSPs interact themselves and become entangled in a network further increasing the
viscosity (Morris and Ris-Murphy, 1981; Malathi and Devegowda, 2001). Because of
the formation of networks with water, water holding capacity of soluble NSPs are
relatively high compared to insoluble NSPs. The insoluble NSPs like cellulose pass
through the gastrointestinal tract unchanged and are biological inert (Annison and Choct,
1991).

The increased viscosity decreases physical contact between endogenous enzymes

and nutrients by acting as a barrier, thus decreased movement of enzymes and substrate
molecules inturn digestibility of starch, proteins, lipids and reduced performance of
broilers. Also increased viscosity reduces rate of passage of digesta and increases
retention time leading to stasis of food for long time, stimulates secretion of digestive
juices more than the required amount thus causes increased endogenous nitrogen loss
besides increasing thickness of unstirred water layer adjacent to intestinal mucosa, this
reduces diffusion of nutrients.

Langout and Schutte (1996) reported that inclusion of high methylated citrus
pectin and low methylated citrus pectin at two levels (1.5 and 3.0%) into the diet had
significantly increased the in vitro viscosity of the diets by 42.7 per cent and water
holding capacity of the diets by 42 per cent.

27
 Channegowda et al. (2001) reported an increased intestinal viscosity with the
increasing levels (10%, 20% and 30%) of sunflower extraction in the broiler diets.
Similarly, Ramesh and Devegowda (2004) recorded an increased intestinal viscosity with
the increasing levels of double zero rapeseed meal in the broiler diet and they concluded
that increased viscosity is mainly due to the increased concentration of soluble NSPs in
the diet. Increased intestinal viscosity was recorded with the feeding of high levels of
sunflower meal and deoiled rice bran in laying hens (Raghavendra, 2003; Shivaramu and
Devegowda, 2004).

2. Alters gut microbial profile

Soluble NSPs increase the viscosity of the digesta, leading to changes in the
physiology and the ecosystem of gut (Choct et al., 1996). This is mainly related to a
slower digesta rate. A slow moving digesta with low oxygen tension in the small
intestine could provide a relatively stable environment where fermentative microflora can
establish (Wagner and Thomas, 1978). The increased bacteria inturn compete for
nutrients with host (Fig 2), cause irritation and thickening of gut mucosa, additionally
increases proliferation of enterocytes, this changes morphology of villi. Increased
viscosity diminishes the function of fatty acids binding protein, which is present in villi
surface.

Choct et al. (1996) demonstrated a large increase in fermentation in the small

intestine of broilers by adding soluble NSP in the diet. At first, it could be thought that
increased production of volatile fatty acids would increase the energy content of the feed,
but due to the drastic change in the gut ecosystem, the net effect was decreased nutrient
digestion accompanied by poor bird performance and subsequent depolymerisation of the
soluble NSP in vivo using glycanases to over come this problem.

28
 Fig 2. Influence of microbial status on AME and BW
of Leghorn chicks
13
106
Mean wt
12.5
104 AME
Mean wt.

AME
12
102
100 11.5

98 11

96 10.5

Germfree Coventional
Germfree Conventional (Bedford, 2000a)

The implication of increased dietary soluble NSP in the onset of other diseases,
such as necrotic enteritis in poultry has also been documented (Kaldhusdal, 1999). The
presence of viscous polysaccharides increases the intestinal microbial activity that are
associated with the bile acid deconjugation, leading to an impaired lipid digestion and
this has been suggested to be partly responsible for poor broiler performance (Choct and
Annison, 1992).

Wagner and Thomas (1978) reported an increased ileal anaerobic counts of

broiler chicks fed diets containing rye or pectin were two or three logarithmic cycles
greater than chicks fed a corn soybean meal diet. Murphy et al. (2004) have also reported
increased number ileal Coliform counts in broilers fed wheat-based diets compared to
control diet.

3. Hinders nutrient digestibility and absorption

It has been clearly demonstrated that presence of NSPs in broiler diets affects the
digestibility of starch, protein and lipids (Devegowda, 1991; Bhat, 1998). Lipid
digestibility is particularly depressed than starch and protein. The probable reason may
be that the increased digesta viscosity resulting from the soluble NSPs may reduce the
constant interaction between potential nutrients and the digestive secretions like
29
enzymes, bile salts etc. In addition, the viscous NSPs are capable of entrapping the bile
salts thereby limiting fat digestion and absorption (Ebihara and Schneenan, 1989).

The end products of digestion, in order to get absorbed must cross an aqueous
barrier, the unstirred water layer, which is adjacent to the intestinal mucosa. The mucus
produced by the goblet cells of intestine participates in the formation of unstirred water
layer by increasing the volume and viscosity adherent to mucosal fluid (Smithson et al.,
1981).
Presence of viscous NSPs induce a secretory response of mucus increase the thickness of
the unstirred water layer thus increasing the resistance for the transport of nutrients
through it adjacent to the epithelial surface. In addition, absorption may be affected by an
increase in proliferation rate of enterocytes and change in the morphology of the villi and
microvilli.
Nagalakshmi and Devegowda, (1992) and Rao and Devegowda, (1996) reported
decreased performance of broilers fed diets containing higher concentration of neutral
detergent fiber and acid detergent fiber. Similarly, Bhat, (1998) reported decreased ME
and crude protein digestibility in broiler chickens with the inclusion of rapeseed meal.

Classen et al. (1985) observed a dose dependent decrease in fat and starch
absorption in three-week-old broiler chicks when fed diets with huskless barley replacing
wheat. Fengler and Morquardt (1988) reported a dose dependent decrease in lipid
digestibility in chicks fed when crude pentosan extracted from rye was added to wheat-
based diet at the rate of 1.5, 3.0 and 6.0 per cent. Klis.et al (1993) studied the effect of
soluble polysaccharides, carboxy methylcellulose on the absorption of minerals from the
gastrointestinal tract of broiler and noted reduced cumulative absorption of minerals with
increasing dietary concentrations of carboxyl methylcellulose. Leske et al. (1993)
reported depressed true metabolizable energy digestibility with the feeding of raffinose
and stachyose from SBM fed to adult birds.

Choct and Annison (1992) observed significant reduction in starch, protein and
lipid digestibility in broiler chickens fed graded levels of wheat pentosans and also
observed that the digestibility of starch, protein and lipid were lower in chicks fed water

30
extracted pentosans than alkali extracted pentosans. Almirall et al. (1993) noted reduced
ileal digestibility of starch, protein and lipids in broiler chicks fed barley-based diets
compared to that in birds fed maize based diets. Similarly, Choct et al. (1996) reported
that addition of soluble NSPs from wheat to broiler diet depressed the ileal digestibility
of starch, protein and lipids by 37.4 and 33.9 and 59.0 per cent, respectively. Silversides
and Bedford (1999) reported an increased intestinal viscosity and reduced apparent
metabolizable energy digestibility in broiler chickens fed wheat based diets

4. Affects the performance of birds

The increase in intestinal viscosity, altered gut microbial profile and reduced
nutrient digestibility and absorption caused by the soluble NSPs leading to decrease in
growth rate and feed efficiency. Several studies have demonstrated these effects and few
of them are reviewed. Classen et al. (1985) reported significant reduction in body weight
of broilers fed varying levels (20, 40 and 60%) of husk less barley.

Choct and Annison (1992) observed depressed AME, growth rate and feed
conversion efficiency of broilers fed sorghum based diet rich pentosans. Inclusion of
graded levels of D-xylose and L-arabinose in broiler diets reduced weight gain, AME,
feed efficiency and dry matter content of droppings (Schutte, 1990). Increased feed
efficiency and litter moisture was reported in laying hens fed diets containing varying
levels of fiber (Mohandas and Devegowda, 1993, Jayanna and Devegowda, 1993 and
Prakash and Devegowda, 1997)

Nagalakshmi and Devegowda, (1992) reported decreased body weight and

increased feed conversion ratio of broiler chickens fed varying levels of crude fiber (12.8,
15.8, 20.7% NDF). Choct et al. (1996) reported reduced AME of the diet, growth rate
and feed conversion efficiency of broiler chickens fed water soluble NSP from wheat.
Langhout and Schuttle (1996) reported significant reduced growth and feed utilisation in
broiler chicks when high methylated pectin was added at 1.5 and 3.0 per cent level.
Suresh and Devegowda, (1996) and Arunbabu and Devegowda, (1997) reported
decreased body weight and feed efficiency in broilers fed diets based on maize, soybean
meal, sunflower meal and deoiled rice bran.

31
 Channegowda et al. (2001) and Kumar, (2002) recorded decreased body weight
and feed efficiency of broilers fed on diets containing sunflower meal and deoiled rice
bran, which are rich in fiber content. Shivaramu and Devegowda, (2004) reported an
increased feed intake and feed conversion ratio in laying hens fed higher levels of
sunflower meal based diets and similarly Raghavendra (2004) reported an increased feed
conversion efficiency with higher levels of de oiled rice bran in laying hens.

2.2.1.3.4. Methods to alleviate the adverse effects

1. Water treatment

The positive effect of water treatment is well established and it is most likely the
result of the removal of water soluble NSPs and the activation of endogenous enzymes
capable of degrading these polysaccharides. The degree of improvement is dependent on
the combination of the water soluble NSPs in the ingredient. This method is found to be
partially effective.

2. Antibiotic supplementation

The response to antibiotic supplementation is also related to the NSP content in

the feed. The probable mechanism of action has been hypothesized as the result of
inhibition and elimination of intestinal microflora, which compete with the host for
available nutrients thus alleviating the antinutritive effect of NSPs.

3. Irradiation

Gamma irradiation has been reported to improve the nutritive value of NSP rich
ingredients, which may be due to its ability to degrade the NSPs with subsequent
reduction in the digesta viscosity.

4. Enzyme supplementation

The enzyme-induced improvement in the nutritive value of poultry diets is well

documented and it is generally conceded that the improvement in performance in relation
to enzyme supplementation is due to the hydrolysis of NSPs and subsequent absorption

32
of the released sugars. Enzymes are proteins with highly complex three-dimensional
molecular structure. They are biological catalysts and act under very specific reaction
conditions (temperature, pH and humidity) only with their specific substrates. They
accelerate the chemical reactions by several folds.

2.3. Feed enzymes

Five types of feed enzymes have been used in the feed industry: NSP-degrading
enzymes, phytate-degrading enzymes, protein-degrading enzymes, starch-degrading
enzymes, and lipid-degrading enzymes. The following section provides a brief overview
of each type of enzyme in terms of their target substrate and mode of action.

2.3.1. NSP-degrading enzymes

Poultry have a limited ability to digest NSP, because of lack of digestive

enzymes. In poultry diets based on ingredients such as wheat, barley, rye or triticale, a
large proportion of the NSP consist of arabinoxylan and β-glucan. NSP-degrading
enzymes (xylanase, β-glucanase and α-galactosidases) have been developed to break
down this type of fibre. The mode of action of NSP-degrading enzymes remains largely
unknown. One or more of the following mechanisms are thought to be involved (Bedford
and Schulze, 1998): (i) Degradation of the NSP in the cell wall matrix of the ingredients
with, the release of encapsulated nutrients, (ii) lowered viscosity of digesta caused by
soluble NSP and improved the rate of diffusion between substrates, enzymes, and
digestion end products, (iii) increased accessibility of nutrients to endogenous digestive
enzymes, (iv) stimulation of intestinal motility and improved feed passage rate, and (v)
supplementation of the enzyme capacity of young animals.

2.3.2. Phytate-degrading enzymes

Phosphorus is stored as phytate (phytic acid) in most plants and therefore is

present in all plant-derived ingredients. About two-thirds of the phytic acid in plant-
33
derived ingredients is not digested or absorbed by monogastric animals. Phytate can also
bind with proteins and minerals to form phytate-protein complexes (Ravindran, 2001).
Phytase can be produced by many species of bacteria, yeast and fungi as well as plants.
However, commercial phytases are commonly produced by Aspergillus niger. Phytase
hydrolyses phytic acid, and liberates phytate-bound P and decreases the need for
inorganic P that is usually added to poultry diets. The effects of microbial phytase on P,
protein, apparent metabolisable energy (AME), and mineral utilisation in poultry and its
mode of action are discussed in the relevant sections of this chapter.

2.3.2. Protein-degrading enzymes

Vegetable proteins contain various anti-nutritional factors. Most legumes contain

lectins and protease inhibitors. Protein inhibitors can cause a reduction in chymotrypsin
activity and impair digestion, while lectins can damage gut wall, impair immune response
and increase endogenous nitrogen loss. Protease works by hydrolyzing proteins or
peptides, and thus improving protein digestibility (Thorpe and Beal, 2001).

2.3.4. Starch-degrading enzymes

The addition of amylase in poultry diets complements endogenous enzymes,

especially in young animals. Amylase can degrade cereal starch to dextrins and sugars,
thereby improving energy availability.

2.3.5. Lipid-degrading enzymes

Use of lipase in broiler diets containing animal and vegetable fats can help the
birds to hydrolyse fats. Thus lipase can improve fat digestibility and enhance energy
utilization in birds.

2.3.6. Sources of feed enzymes

Various fungi, bacteria and to some extent yeast are employed in enzyme
production. It is essential for these microorganisms to produce enzymes as they sustain

34
their own viability by producing enzymes to breakdown substrates for further
metabolism. Fungi represent largest group among enzyme producing microorganisms.
The most important genera are Aspergillus sp., Trichodema Sp., Penicillin sp. And
Hemicela sp. Among bacteria Bacillus subtilis and Bacillus lichaniformis.

2.3.7. Production of enzymes

There are mainly two ways of production of enzymes on an industrial scale. They
are,

2.3.7.1. Submerged liquid fermentation

Submerged fermentation involves submersion of the microorganisms in an

aqueous solution containing all the nutrients needed for growth.

Commonly used enzymes in animals nutrition

Enzyme Substrate
Cellulase Cellulose
Pentosanase (xylanase) Pentosans (arabinoxylase)
-glucanase -glucans
pectinase Pectins
amylase Resistant starch
protease Proteins
-galactosidase Oligosaccharides(-galactosides)
Phytase Phytic acid / phytates
Tannase Tannins

2.3.7.2. Solid substrate fermentation (SSF)

Solid substrate fermentations are generally characterized by growth of

microorganism on water insoluble substrates in the presence of varying amounts of free
water. This process is also known as solid-state fermentation

2.3.7.3. Differences in enzymes produced by SSF and submerged liquid fermentation

35
 The enzymes produced by SSF technology were found to be more resistant to
heat denaturation and contained more mixtures of enzyme activities than those produced
in submerged liquid fermentation by the same strain.

Ayers et al. (1952) reported that pectinases produced by SSF had noticeable
biochemical differences from those produced by submerged fermentation. A glucosidase
produced by Aspergilus phoenicis in SSF was more thermotolerant than when produced
in submerged liquid fermentation (Deschamps and Huet, 1984). Alazard and Raimbault
(1981) showed that amylases produced by A. niger using SSF were more resistant to heat
denaturation than those produced in submerged liquid fermentation by the same strain.

Exoenzyme production in SSF system results in increased amounts of some

enzymatic activities not produced by cultures in liquid fermentation. A phytase produced
by SSF also contains a mixture of activities not found in enzyme mix from submerged
culture systems. The complex nature of feedstuffs makes there side activities beneficial
to the animal industry (Classen, 1996).
In vitro comparisons have shown increased rates of reducing sugar and amino nitrogen
and an associated increase in phosphate release by an SSF phytase product (Filer et al.,
1999).
Comparison of enzyme activities of two commercial available phytases
Production method
Enzyme assayed
Submerged liquid SSF
Phytase 1500 PU/g 1500 PU/g
Fungal -amylase Below detectable level 240 FAU/g
-glucanase Below detectable level 2160 BGU/g
Cellulase Below detectable level 310 CMCU/g
Fungal protease Below detectable level 7380 HUT/g

2.3.8. Effect of enzymes on performance of broilers fed corn-soy based diets

Published literature on the effects of enzyme on the performance of broilers fed corn-
soybean meal based diets is summarized in the Table 9, 10 and 11. The addition of
enzymes to corn-soybean meal based broilers diets have been found to improve weight
36
gain and feed intake of broilers by 0.25 to 8.13% and –7.41 to 5.80 %, respectively. Feed
conversion ratio lowered by 0.10 to 3.8%.

2.3.9. Effect of enzymes on intestinal viscosity

Classen et al. (1996) observed that increased viscosity of the digesta was mainly
due to soluble NSPs (pentosans), which acts as a barrier between endogenous enzymes
and nutrients. Additionally, increases moisture of droppings leads to not only
environmental pollution by release of ammonia, but also cause hock and breast damage
of birds.

Choct et al. (1996) observed an increased gut viscosity, reduced AME and feed
efficiency of broilers with the addition of soluble NSPs (extracted from wheat) to the diet
and they reported that enzyme supplementation reversed the adverse effects by
improving weight gain, AME and feed efficiency. Bedford et al. (1991) reported that
pentosanase supplementation to rye based broiler diets improved weight gain and feed
efficiency and reduced foregut and hindgut viscosity and they stated that improved
weight gain and feed efficiency was correlated much with foregut viscosity rather than
hindgut viscosity.

37
 Table 9. Effect of enzyme complex on performance of broiler chickens
References Diet type Enzymes Body weight Feed intake FCR
gain (%) (%) (%)

Nagalakshmi and Corn-soy- Beta-glucanase +9.04 - -3.2

Devegowda. (1992) GNC Cellulase
Hemicellulase

Marsman et al. (1997) Corn-soy Protease +1.05 +0.9 0

Carbohydrase

Graham (1996a) Corn-soy Xylanase +2.5 - +3.6

Protease
Amylase

Graham (1996b) Corn-soy Enzyme complex +1.33 -2.34 -3.62

Suresh and Devegowda. Corn-soy-SFE Beta-glucanase +2.1 +2.3 +1.2

(1996) Cellulase
Hemicellulase

Swift et al. (1996) Corn-soy Protease (24,500 HUT/g) +1.79 -7.41 -2.63
(pelleted) Amylase (6700 FUA/g)
Pentosanase (1000 XU/g)
Cellulase (250 CMCU/g)

Schang et al. (1997) Corn-soy Protease (24,500 HUT/g) +4.7 +3.2 -1.3
Amylase (6700 FUA/g)
Pentosanase (1000 XU/g)
Cellulase (250 CMCU/g)
-galactosidase

38
 Table 10. Effect of enzyme complex on performance of broiler chickens

References Diet type Enzymes Body weight Feed intake FCR

gain (%) (%) (%)

Bhat. (1998) Corn-soy-SFE Avizyme 1500 +5.02 +2.23 -2.58

Zanella et al. (1999) Corn-soy Xylanase (800 U/g) +1.9 - -2.2

Protease (6000 U/g)
Amylase (2000 U/g)

Channegowda et al. Corn-soy Xylanase +6.60 +2.80 +1.0

(2001) Pectinase
Beta-glucosidase

Roy et al. (2003) Corn-soy -galactosidase (20 IU/kg) +4.85 +2.66 -1.90
Cellulase (560 IU/kg)

Graham et al. (2002) Corn-soy -galactosidase 2.63 3.85 -1.21

Kocher et al. (2002) Corn-soy Endo-1,3(4)--glucanase (54.7 +0.25 -0.88 -1.16

FBG/g)
Hemicellulase (15000 UHCU/g))
Pectinase (3017 U/g) (2.0 g/kg)

Kumar. (2002) Corn-soy Xylanase +3.77 +2.3 +1.86

Pectinase
Cellulase

39
 Table 11. Effect of enzyme complex on performance of broiler chickens

References Diet type Enzymes Body weight Feed intake FCR

gain (%) (%) (%)
Rao et al. (2003) Corn-soy Amylase (1008 SKBU/kg) +8.13 +4.72 -0.54
Xylanase (1040 U/kg)
Protease (1379 HUT/kg)
Phytase (77 FTU/kg)
Cellulase (3000 CMU/kg)

Kocher et al. (2002) Corn-soy Xylanase (800 U/g) + - +4.28

Protease (60000 U/kg
Amylase (2000 U/kg)

Kocher et al. (2003) Corn-soy -glucanase (54.7 FBG/g + - +0.10

Hemicellulase (15000 UHCH/g)
Pectinase (3,017 U/g)

Rani et al. (2003) Corn-soy -amylase +0.59 - -0.18

Cellulase
Xylanase
Pectinase

Jackson et al. (2004) Corn-soy -mannase (80 or 110 MU/ton) +4.8 -1.11 -3.8

Dalibard et al. (2004) Corn-soy Enzyme mix (Rovadio Excel) +1.6 - -2.5

Ramesh and Corn-soy Xylanase +1.93 +3.37 -

Devegowda. (2004) Pectinase
Cellulase

40
Bedford and Classen (1993) identified foregut digesta viscosity as a major determinant
on performance of broilers fed wheat-barley based diets and reported soluble and high
molecular weight polysaccharides are responsible for high digesta viscosity, which
resulted in poor feed efficiency and depressed the body weight. Crouch et al. (1997)
evaluated the efficiency of xylanase (1000 U/g) supplementation to 40 per cent wheat
and corn-soy based diets and concluded that enzyme supplementation had lowered the
intestinal viscosity and improved the performance of chicks.

Dusel et al. (1998) reported significant reduction in intestinal viscosity, improved

AME and nutrient digestibility with the addition of xylanase in broiler fed wheat based
diets. Silversides and Bedford (1999) studied intestinal viscosity, AME and performance
of broilers fed on wheat based diets and found that intestinal viscosity accounted for 50
to 90 per cent of the variation of AME, indicating increased viscosity due to soluble
NSPs of wheat reduces the availability of energy.

Channegowda et al. (2001) conducted a trial of six-week duration to determine

the effects of two enzyme mixtures (A and B) on sunflower extraction (SFE) based diets
fed broilers. Birds were fed on diet containing 0, 10, 20 per cent SFE supplemented with
or without enzyme A (1 kg/ton) or B (2 kg/ton). The relative viscosity of intestinal
contents was increased in parallel with SFE levels. Addition of enzyme A or B reduced
the digesta viscosity considerably, but did not improve performance.

Malathi and Devegowda (2001) conducted a two-stage in-vitro digestion assay to

evaluate the NSP digestibility of different feed ingredients by enzymes and digestibility
was assessed by measuring the relative viscosity. They reported that addition of enzymes
(xylanase, pectinase and cellulase) significantly reduced the relative viscosities of the
digesta after first phase of incubation.

Kumar (2002) recorded decreased intestinal viscosity with the supplementation of

enzymes (xylanase, cellulase and pectinase) in broilers fed diets containing varying levels
of full fat deoiled rice bran. Channegowda et al. (2001) observed similar results in

41
broilers fed sunflower meal and Shivaramu and Devegowda (2004) and Raghavendra.
(2003) in laying hens fed varying levels of sunflower meal.

Zenella et al. (1999) and Kocher et al. (2002) did not notice any change in
intestinal viscosity of broilers chicken fed on corn-soy diets with the enzyme addition.
Kocher et al. (2000) reported similar results in broilers fed sunflower meal and rapeseed
meal with enzyme addition. Ramesh and Devegowda (2004) recorded reduced intestinal
viscosity with enzyme supplementation (xylanase, pectinase, cellulase) in broilers fed
double zero rapeseed meal.

2.3.10. Effect of enzymes on gut microflora of broiler chickens

Overall responses to the enzyme supplementation in growing birds are variable.

Factors affecting enzyme response in wheat-based diet include variability in added
enzymes, quality of ingredients, breed and age of birds (Bedford, 1996). Some evidence
also suggests that the enzyme response may be related to gut microflora of birds and
microbial interactions (Choct et al., 1996, Apajalathi and Bedford, 1999).

It has been suggested that the increased digesta viscosity, caused by high levels of
soluble NSP may stimulate the growth of anaerobic microflora and their interaction with
nutrients. The anaerobic microflora that are generally found in large numbers in the
cecca, also tend to migrate to small intestine where most nutrient absorption takes place
(Campbell and Bedford, 1992). Those unbeneficial bacteria can irritate the gut lining and
result in a thicker lining with damaged microvilli. It has been shown that enzyme
supplementation appears to reduce the microbial population in the intestinal tract (Choct
et al., 1996, 1999, Sinale and Choct, 2000) and thus the negative effects of NSP on
intestinal villi.

Choct et al. (1999) reported that supplementation of xylanase to wheat based diets
reduced the microbial activity in the ileal digesta as indicted by reduced concentration of
volatile fatty acids. Sinale and Choct (2000) reported that addition of xylanase to a
wheat-based diet reduced the number of undesirable organisms such as clostridium
pefringens in the caeca.

42
 Choct and Annison (1992) reported that NSPs could increase the viscosity of the
intestinal contents, thereby affecting nutrient absorption. Furthermore, the finding that
dietary addition of antibiotics improves the performance of chickens fed cereal-based
diets, especially those containing rye, has led to the suggestion that the anti-nutritional
effect of NSP is directly or indirectly mediated by the gut microflora. Engberg et al.
(2004) reported that xylanase addition stimulates growth of lactic acid bacteria in the
small intestine, which was confirmed by higher lactic acid concentration and higher ATP
concentration at the location. However, they reported that the concentrations of other
shorter chain fatty acids, in particularl acetic acid, in the small intestine were not
influenced by xylanase supplementation. Engberg et al. (2004) in the same study
reported the significant decrease in the cecal pH in the birds supplemented with xylanase.
It has been suggested that sugars such as xylose and xylo-oligomers that escape
enzymatic digestion may enter cecum and may be fermented by cecal microflora, which
contributed for the decrease in pH.

Murphy et al. (2004) reported that xylanase addition to wheat based broiler diets
resutled in a significant increase in the coliform count in the crop and no significant
difference between diets in the lactic acid bacterial counts in the crop or ileum.
However, the ileal counts tended to be numerically lower for the diets treated with the
new xylanases suggesting that, reductions in intestinal viscosity and the subsequent
increase in absorption, results in reduced bacterial colonisation through substrate
limitations. Apajalathi and Bedford (1999) reported that addition of enzyme increased
the number of the species of Peptostreptococcus, Bacteriodes, Propionibacterium,
Subacterium and Bifidobacterium and decreased the number of the species of
Clostridium, Enterobacteriacaea and Campylobacter. Lactic acid bacteria as a group is
unaffected in this study, however they have reported an increased lactobacilli numbers by
inclusion of xylanase in wheat based diets. A same author investigated the effects of
enzyme addition on an ileal microfloral population of broiler chickens and he stated that
addition of enzyme has improved the digestibility of the ration as far as chick is
concerned, which results in these being less substrate (starch, protein and fat) available
for the bacterial community. As a result, use of enzyme reduced ileal population
significantly. Such a significant reduction in bacterial number not only results in terms of
43
competition for nutrient but also reduces the likelihood of a critical threshold being
crossed.

Research conducted by Tan and Hruby (2003) in the United Kingdom has shown
that the use of feed enzyme significantly reduced the number of food poisoning bacteria
in broilers. These authors conducted several trials to evalute the effect of enzymes on the
cecal microflora population and they reported that in the eight wheat based trials, there
was two third reduction in the number of campylobacter found in birds fed the enzyme
supplemented diet and in the four corn based trials there was a reduction of over a third
in birds fed the enzyme treated diet, compared with the control. In the three corn based
trials, there was a reduction of almost 60 per cent in the number of Salmonella found in
birds fed the enzymatic treated diet and a significant reduction in the number of
Salmonella found in birds fed the enzyme treated wheat based diets.

The reduction of microflora populations with addition of enzyme appears to be

linked to three key modes of action. (1) A reduction in intestinal viscosity associated with
wheat results in increased feed passage rate, which means that there is less substrate
available to support the harmful bacteria. (2) An increased in nutrients digested by the
bird – results in fewer nutrients for the growth of harmful bacteria. (3) An altered
carbohydrate profile in the intestine – results in more of substrate preferred by beneficial
bacteria eg., Lactobacillus.

So, the effect of these enzymes is to reduce the amount of substrate available for
the development of potentially harmful bacteria in the gut.

2.3.11. Effect of enzymes on nutrient digestibility of broilers

The effect of enzymes in improving the apparent metabolizable energy (AME) in poultry
fed corn-soy based diets has been demonstrated in few recent studies in broilers (Pack
and Bedford, 1997, Bhat, 1998, Zanella et al., 1999, Graham et al., 2002, Kocher et al.,
2002, 2003). Overall, the addition of enzymes to broiler diets based on corn-soy
increased in the AME by 0.52 to 11.9% (Table12 and 13).

44
 Nagalakshmi and Devegowda, (1992) and Rajeshwara Rao and Devegowda,
(1996) reported an improved digestibility of neutral detergent fiber and acid detergent
fiber with the supplementation of enzyme complex containing

45
 Table 12. Effect of enzyme complex on apparent metabolizable energy in broiler chickens
References Diet type Enzymes AME
Improvement
(%)
– +
Charlton (1996) Corn-soy Protease - - +4.2
Amylase
Pentosanase
Cellulase
-galactosidase
(Allzyme vegpro)

Swift et al. (1996) Corn-soy Protease 68.9 71.5 +2.6

(pelleted) Amylase
Pentosonase
Cellulose
-galactosidase
(Allzyme vegpro)

Pack and Bedford Corn-soy Amylase 2907 2971 2.2

(1997) Xylanase
Protease

Bhat. (1998) Corn-soy Avizyme 1500 2838 2909 2.5

Zanella et al. (1999) Corn-soy Xylanase (800 U/g) 2580 3658 2.18
Protease (6000 U/g) (ME Kcal/g)
Amylase (2000 U/g)

Graham et al. (2002) Corn-soy -galactosidase 2974 3328 11.9

Table 13. Effect of enzyme complex on apparent metabolizable energy in broiler chickens

46
 References Diet type Enzymes AME Improvement
(%)
+ _
Kocher et al. (2002) Corn-soy Endo%3(4)--glucanase 3068 3116.56 1.58
(54.7 FBG/g)
Hemicellulase (15,000
UHCU/g)
Pectinase (3017 U/g)

Carbohydrase (- 3068 3118.95 1.66

galactomanase)
(0.94 g/kg)

Kocher et al. (2003) Corn-soy Xylanase (800 U/g) 3040 3056.00 0.52
Protease (6000 U/g)
Amylase (2000 U/g)

-glucanase (54.7 FBG/g) 3040 3116.56 2.52

Hemicellulase (15,000
VHCH/g)
Pectinase (3,017 U/g)

47
 the activities of cellulase, hemicellulase, beta-glucanase and xylanase, amylase,
protease, respectively, in the broiler diets.

Bhat, (1998) reported an improved ME with the inclusion of Avizyme 1500 to

corn-soy diet (2909 Vs 2838) and Avzyme 1500 to rapeseed meal diet (2925 Vs 2852) of
broiler chickens. Similarly, Devegowda (1996) reported an improved nutrient
digestibility with the supplementation of enzymes in broiler.

The published information on the effects of enzyme on protein utilization in

broilers fed corn-soy diets is limited. The effects of enzymes on protein utilization in
broilers fed corn-soy diet have been examined by Graham. (1996), Swift et al. (1996),
Pack and Bedford. (1997), Marsman et al. (1997), Bhat. (1998), Zanella et al. (1999), and
Kocher et al. (2002). Improvements in digestibility have been reported in most studies. In
general, the improvement in nitrogen digestibility with addition of enzyme in poultry fed
corn-soy diet, ranging from 1.5 to 9.5%(Table 14).

2.3.12. Effect of enzymes on mortality

Pack and Bedford (1997) pooled the research observation of effect of enzymes on
mortality from 51 growth trials conducted at different research institutions in different
husbandry conditions and reported a substantial reduction in bird mortality in the groups
fed the enzymes up to 19 per cent. Similarly, Jackson et al. (2004) reported reduction in
mortality by 43 per cent in the birds fed

48
 Table 14. Effect of enzyme complex on apparent ileal digestibility of protein in broiler chickens
* Nitrogen digestibility
References Diet type Enzymes Ileal digestibility Improvement
(%)
– +
Swift et al. (1996) Corn-soy Allzyme vegpro 24.3* 33.8 +9.5
(pelleted)

Graham (1996b) Corn-soy Protease 79.5 84.9 +5.4

Marsmann et al. (1997) Corn-soy Protease 83.7 85.2 +1.5

Carbohydrase

Pack and Bedford (1997) Corn-soy Amylase 80.0 82.0 +2.0

Xylanase
Protease

Bhat. (1998) Corn-soy Avizyme 1500 65.71 66.43 +1.1

Zanella et al. (1999) Corn-soy Xylanase (800 U/kg) 70.9 73.6 +2.7
Protease (6000 U/kg) (1 g/kg)
Amylase (2000 U/kg)

Kocher et al. (2002) Corn-soy Endo 1,3(4)--gluconase (54.7 FBG/g) 82.0 86.0 +4.87
Hemicellulose (15000 VHCU/g) (2
g/kg)
Pectinase (30 HU/g

Carbohydrase (-galactonase) 82.0 81.0 -1.2

(0.94 g/kg)

49
corn-soy diet with the enzyme -manase and also Rajamane (1992) recorded higher
survivability in broilers fed diets with enzyme than their controls.

2.3.13. Effect of enzymes on dressing percentage

Zanella et al. (1999) and Roy et al. (2003) reported that, addition of enzymes to
corn-soy diets had no effect on dressing percentage of broiler chickens and the mortality
was negligible in all the groups.

2.4. Phytate phosphorus

Most of plant seeds contain more than 60 to 80 per cent of phosphorus in phytate
form (phytic acid), which is not available to the bird (Ravindran et al., 1995). The phytic
acid also exerts a negative influence on the solubility of proteins and function of pepsin.
The phosphate groups of the inositol ring can bind various cations like calcium,
magnesium, iron and zinc in a fixed complex and thus interfere with their availability.
Supplementation of phytase enzyme in the feed releases the bound phosphorus and other
cations.

Figure 3. Structure of phytic acid

As seen in Figure 3, phytate bears six P groups on one 6-carbon molecule. At neutral pH
the phosphate groups in phytic acid have either one or two negatively charged oxygen
atoms (Reddy et al., 1982). Therefore, various cations can chelate strongly between two
phosphate groups or weakly with a single phosphate group. As a result, phytic acid can
bind mineral elements and amino acids, and reduce their bioavailability.

50
2.4.1. Phytate Phosphorus Concentration in Plant-Derived Ingredients
The levels of phytate P in common feedstuffs have been reported by a number of
workers (Nelson et al., 1968a, Reddy et al., 1978, Ravindran et al., 1994, Devegowda,
2005). Compilation of phytate P in common feedstuffs are also available (Reddy et al.,
1982, Ravindran et al., 1995). Typical levels of phytate P of common feedstuffs are
shown in Table 15 and 16.
The level of phytate P in a feedstuff generally depends on the part of the plant from
which it is derived. In general, oilseeds meals and cereal by-products contain large
amounts of phytate P, whereas, cereals and grain legumes contain only moderate amounts
(Ravindran et al. 1995). The proportion of phytate P varies from 60-80% of the total P in
seeds of cereals, grain legumes and oil-bearing plants. In most cereals, phytic acid is not
uniformly distributed within the kernel, but associated with specific morphological
components of the seed. Phytate concentration in plant materials depend on several
factors including the stage of maturity, degree of processing, cultivar, climate, water
availability, soil, geographical location and year (Reddy et al., 1982).

Table 15. Phosphorus content in plant materials

Ingredients Total P (%) Phytate P (%) Avail P (%)

Soybean meal 0.65 0.38 0.27
Groundnut extraction 0.68 0.49 0.19
Sunflower extraction 0.92 0.62 0.30
Fish meal 2.00 - 2.00
Meat meal 4.00 - 4.00
Dicalcium phosphate 16.18 - 16-18
Maize 0.36 0.23 0.13
Rice broken 0.32 0.28 0.04
Bajra 0.65 0.41 0.24
Rice polish 1.25 1.10 0.15
De oiled rice bran 1.35 1.16 0.19
(Devegowda, 2005)

51
 Table 16. Phytate phosphorus (P) content in various feed ingredients

Phytate P Phytate P
Ingredient
g/100 DM (as % of total P)
Cereals
Barely 0.27 94
Corn 0.24 72
Rice (polished) 0.27 77
Sorghum 0.24 66
Wheat 0.27 69
Cereal by-products
Rice bran 1.03 80
Rice polishings 2.04 89
Wheat bran 0.92 71
Grain legumes
Field peas 0.24 50
Oilseed meals
Rapeseed meal 0.70 59
Sesame meal 1.02 81
Soybean meal 0.39 60
Sunflower meal 0.89 77

(Ravindran et al. 1995)

52
2.5. Phytases
The phytate can be hydrolysed by phytases. There are three sources of phytase
namely, plant phytase, intestinal phytase and microbial phytase.
2.5.1. Plant Phytase
Endogenous phytase activity in feedstuffs is variable (Table 3). The highest
activities are reported in rye, wheat and wheat bran (Ravindran et al., 1995a). In contrast,
corn, sorghum and oilseeds have very little endogenous phytase activity. Published data
on the effects of plant phytase activity on animal performance is limited.
2.5.2. Intestinal Phytase Activity
The presence of intestinal phytase activity in poultry is controversial. Liebert et
al. (1993) reported that the phytase activity in the contents of the crop, stomach and small
intestine of chickens is negligible. Kornegay and Yi (1999) stated that the significance of
phytase produced by microorganisms residing in the intestinal tract is negligible. Maenz
and Classen (1998), however, reported that intestinal brush border alkaline phosphatase
could contribute to degradation of phytate P. The specific and total activities of alkaline
phosphatase in intestinal brush border were highest in the duodenum and declined in the
jejunum and ileum. When 4-wk-old broilers and laying hens were compared, the specific
activity of alkaline phosphatase were comparable, but the hens had a 35% higher total
activity of alkaline phosphatase in the brush border.

2.5.3. Microbial Phytase

Microbial phytase can be found in numerous bacteria, yeast and fungi.
Aspergillus is the most widely used fungi in the commercial production of microbial
phytase.
2.5.4. Factors that affect phytate p utilisation
The ability (or inability) of chickens to utilise phytate P has been reviewed by
Nelson (1967), and Ravindran et al. (1995). Phytate P utilisation by poultry is influenced
by several factors including dietary levels of calcium (Ca), P, ratio of Ca to total P, levels
of vitamin D3, age and genotype of the animal, citric acid, dietary fibre and size of feed
particles. Phytate P utilisation in poultry has been shown to range from 10 to 87%
depending on dietary levels of Ca, P, vitamin D3, microbial phytase, and diet type

53
(Edwards, 1993). The following sections provide an overview of the abovementioned
factors.
1. Ca, P and Ratio of Ca to Total P
Dietary levels of Ca, P, and the ratio of Ca to total P are the major factors that
affect phytate P utilisation, with the effects of dietary Ca being much greater (Ravindran
et al., 1995). Two possible mechanisms for the decreased phytate P utilisation at high
dietary Ca levels have been proposed (i) the precipitation of phytate by Ca through the
formation of extremely insoluble Ca-phytate complexes that are less accessible to
phytase, (ii) the direct depression of phytase activity resulting from extra Ca competing
for the active sites of phytase. In addition to dietary Ca and P levels, the ratio of Ca to
total P plays an important role in phytate P utilisation. A Ca to total P ratio between 1.1
to 1.4 appears to be optimal for phytase action (Qian et al., 1997). This was further
confirmed by Zyla et al. (2000) who reported that weight gain, feed intake, and toe ash of
broilers fed diets supplemented with phytase were negatively affected when the dietary
ratio of Ca to total P was increased from 1.44 to 1.93. These adverse effects were
attributed to the formation of Ca-phytate complexes that are resistant to phytase action.
2. Vitamin D3
The interaction of dietary vitamin D3 with both dietary Ca and P levels can affect
phytate P utilisation. When birds are fed diets marginal or deficient in vitamin D3,
phytate P utilisation is depressed (Ewing, 1963). Applegate et al. (2000) reported that
apparent ileal phytate P digestibility was increased from 42.9 to 64.0% by 1,25-(OH)2D3
supplementation. Phytate P utilisation in response to vitamin D3 supplementation is
related to dietary levels of Ca and P. Mohammed et al. (1991) reported that a decrease in
dietary calcium improves phytate digestibility in broilers. Phytate P digestibility was 50%
when the diet contained 1.0% calcium, 0.69% total P, and 12.5 µg/kg 1,25-(OH)2D3.
The utilisation increased to 77% when both levels of Ca and total P in the diet were
decreased to 0.5%. The mechanisms of improved phytate P in response to vitamin D3
supplementation are unknown. However, one or more of the following may be involved
(Ravindran et al., 1995): (i) increased synthesis or activity of intestinal phytase, (ii)
increased phytate hydrolysis by stimulation of calcium absorption, thus rendering the
phytate more soluble and available for utilisation, or (iii) enhanced absorption of P.

54
3. Age and Genotype of Birds
It is generally accepted that older birds hydrolyse more phytate P than chicks, as
there is more dephosphorilating activity present in the gastrointestinal tract of older birds
(Ravindran et al., 1995). The ability of poultry to utilise phytate P also increases with
age.
2.5.5 Effect of Microbial Phytase on Bird Performance and Bone Mineralisation
The effects of microbial phytase on the performance, bone mineralisation and P
utilisation in various poultry species are summarised in Tables 17 and 18. The addition of
microbial phytase (500-750 FTU/kg) to low-P corn-based diets have been found to
improve weight gain and feed intake of broilers by 5.9-99.7%, and .1-59.1%,
respectively. Feed conversion ratio (FCR) values can be lowered by 1.9-16.2%.
Improvements in feed efficiency with phytase addition have been reported in a number of
studies but not in others (Denbow et al., 1995, Kornegay et al., 1996, Sebastian et al.,
1996, Ravindran et al., 1999, Jayashree, et al., 2001). The influence of microbial phytase
on bone mineralisation has been consistent in both corn- and wheat-based diets. Addition
of phytase to low-P diets improved bone mineralisation to levels comparable to those of
adequate-P diets in growing birds (Tables 17 and 18) and layers (Klis et al., 1997, Carlos
and Edwards, 1998).

2.5.6. Effect of Microbial Phytase on P Digestibility and Utilisation

The beneficial effect of supplemental phytase on P availability, measured as bone
ash, in broilers was first reported by Nelson (1971). The effectiveness of microbial
phytase in improving phytate-bound P availability, P retention and reducing P excretion
has been demonstrated in studies with broilers (Table 17), Addition of microbial phytase
to broiler diets (500-750 FTU/kg) increased apparent P retention by 3.1-12.5 percentage
units. When compared to adequate-P (0.45-0.47% non-phytate P) diets, addition of
microbial phytase to low-P (0.15-0.37% non-phytate P) broiler diets reduces P excretion
by 20-57% (Table 17).
2.5.7. Effect of microbial phytase on the utilisation of Nutrients other than
phosphorus
Phytate-Protein Complexes
55
 Phytic acid is generally considered as a factor primarily limiting P availability
from plant derived materials. However, phytate can also form phytate complex with
protein and a range of minerals. In the highly acidic stomach region, amino acids, in
particular lysine (Lys), methionine (Met), arginine (Arg) and histidine (His) can bind
directly to phytate P creating insoluble phytate-protein complexes. In the less acidic
region of the intestine, mineral cations (Ca, Mg, Zn, Fe) act as a bridge between phytate
P and protein, resulting in protein-mineral-phytate complexes. Phytate-protein complexes
are insoluble and less subject to attack by proteolytic enzymes than the same protein
alone (Anderson, 1985).
In vitro studies have shown that phytate may form complexes with proteins and
free amino acids. Jongbloed et al. (1997) reported that soluble proteins in casein, corn,
rice polishings, soybean meal and sunflower meal were substantially precipitated in the
presence of phytic acid. Incubation of feedstuffs with microbial phytase largely prevented
the precipitation.

56
 Table 17. Effects of phytase on bird performance, bone mineralization, and phosphorus (P) utilization in broiler chickens

Phytase NP Weight Feed intake1 1

Toe ash1 P retention1
References Diet type FCR
(FTU/kg) (%) gain1 (%) (%) (% units) (% units)
Simons et al. (1990) Corn-soy 500 0.15 + 84.3 - -15.7 - +9.8
750 0.15 + 99.7 - -16.2 - +9.8
Pernery et al. (1993) Corn-soy 500 0.32 + 12.5 +8.6 -3.8 +1.2 +8.0
Denbow et al. (1995) Corn-soy 600 0.20 + 70.9 +59.1 -10.9 +2.3 -
600 0.27 + 36.9 +39.0 +2.0 +1.6 -
600 0.30 + 13.6 +13.1 - +1.1 -
Kornegay et al. (1996) Corn-soy 600 0.20 + 37.9 +40.2 -2.3 +1.6 +3.1
0.27 + 10.4 +10.7 - +1.5 +8.7
0.34 + 5.9 +4.1 -1.9 +0.5 +6.3
Sebastian et al. (1996) Corn-soy 600 0.33 + 13.3 +13.5 - - +12.5

Camden et al. (2001) Corn-soy 500 0.28 + 12.1 +8.0 -3.4 +0.8 +4.6
Jayashree et al. (2001) Corn-soy 1000 0.27 +2.10 +4.30 - - -
438 0.27 +5.00 +5.8 - - -
1150 0.27 +10.03 +7.5 -4.01 - -
1
Percentage changes for phytase addition diets over the low-P basal diets;
nP=non-phytate P

57
 Table 18. The effects of combination of microbial phytase with other enzymes on the performance
Weight Feed Feed/grain1 AME1
References Treatment Diet type
gain1 (%) intake1 (%) (%) (%)
Simbaya et al. (1996) B+phytase Wheat-canola meal +1.0 - -2.6 -

B+phytase +6.3 - -7.2 -

+carbohydrase+protease
Ravindran et al. (1999) B+phytase Wheat-soy - - - +4.5

B+phytase +xylanase +5.0 +1.6 -3.2 +6.6

Zyla et al. (1999) B+phytase Wheat-soy +9.6 +5.0 -4.4 -

B+phytase +xylanase +21.5 +8.3 -10.8 -

Zyla et al. (2000) B+phytase Wheat-soy +13.4 +7.4 -5.1 -

B+phytase +xylanase +26.4 +18.1 -6.3 -

Selle et al. (2001) B+phytase Wheat-soy +3.8 -3.0 -6.9 -

B+phytase +xylanase +6.7 -3.0 -9.4 +2.8
1
Percentage changes over the low-P unsupplemented basal diet
B=basal diet

58
 Phytate can inhibit a number of digestive enzymes such as pepsin, α-amylase and
trypsin (Ravindran, 2001). The inhibition of digestive enzymes by phytate appears to
involve non-specific protein binding. Inhibition may also result from the chelation of
calcium ions that are essential for the activity of trypsin and α-amylase, or possibly from
an interaction with the substrates used by these enzymes (Ravindran, 2001). Phytate can
inhibit a number of digestive enzymes such as pepsin, α-amylase and trypsin (Ravindran,
2001). The inhibition of digestive enzymes by phytate appears to involve non-specific
protein binding. Inhibition may also result from the chelation of calcium ions that are
essential for the activity of trypsin and α-amylase, or possibly from an interaction with
the substrates used by these enzymes (Ravindran, 2001).
2.5.8. Effect of Microbial Phytase on Protein Utilisation
Influence of microbial phytase on protein/amino acid digestibility in broiler
chickens (Ravindran et al., 2000, Ravindran et al., 2001), has extensively been examined.
In general, the improvement in nitrogen digestibility by addition of microbial phytase in
poultry diets is small, ranging from 0.9 to 4.9% (Table 19).
2.5.9. Effect of Microbial Phytase on Energy Utilisation
An “ energy effect “ for phytase addition to broiler diets was first reported by
Rojas and Scott (1969). In this study, the AME contents for chickens fed cottonseed meal
and soybean meal following treatment with a crude phytase preparation from Aspergillus
ficuum were significantly improved. Using a similar crude enzyme product, Miles and
Nelson (1974) also observed that there was a significant improvement in the AME
content for chickens fed phytase-treated

59
 Table 19. The effect of phytase on apparent ileal digestibility of
nitrogen (N) in broiler chickens

Phytase nP1 N improvement

References
(FTU/kg) (%) (%)

Sebastian et al. (1997) 600 0.31 4.9

Namkung and Leeson (1999) 1200 0.35 3.2
Ravindran et al. (2000) 400 0.23 3.6
800 0.23 3.0
Ravindran et al. (2000) 400 0.45 2.2
800 0.45 2.4
Camden et al. (2001) 500 0.30 2.3
Ravindran et al. (2001) 500 0.45 4.0
750 0.45 3.7
1
non-phytate P

60
cottonseed meal (7.1 vs 8.5 MJ/kg) and wheat bran (4.6 vs 7.0 MJ/kg), but not for
soybean meal (11.5 vs. 10.7 MJ/kg). It is, however, possible that the energy responses
observed in these studies may have been partly due to the presence of other enzymes in
the crude preparations. The effect of microbial phytase in improving AME in poultry fed
corn-, wheat- and sorghum-based diets has been demonstrated in a number of recent
studies in broilers (Ravindran et al., 2000). Overall, the addition of phytase to poultry
diets increased the AME by 1.1-6.3% depending on dietary level of non-phytate P and
diet type (Table 20). The mode of action underlying the effect of microbial phytase on
energy utilisation is not fully understood. Several possible mechanisms have been
proposed (Ravindran, 1999). Firstly, improvements in protein and amino acid utilisation
with added phytase may contribute, at least in part, to the observed energy effects.
Secondly, a wide ratio of Ca to total P leads to the formation of insoluble Ca-phytate
complexes. The latter complexes can further react with fatty acids in the gut lumen to
form insoluble metabolic soaps thereby lowering fat digestibility. Microbial phytase may
prevent the formation of the insoluble Ca-phytate complexes by hydrolysing the phytate.
It is also possible that phytase may reduce the adverse effects of phytic acid on starch
digestion and endogenous losses.
2.5.10. Effect of Microbial Phytase on Ca Utilisation
Supplementation of microbial phytase in poultry diets has been shown to improve
Ca availability and retention in broilers (Simons et al., 1990, Sebastian et al., 1996,
Kornegay and Yi, 1999 and Jayashree et al., 2001). Simons et al. (1990) reported that the
addition of 500 FTU/kg microbial phytase to a low-P diet (0.15%

61
Table 20. The effect of phytase on apparent metabolizable energy (AME; MJ/kg dry matter) in poultry

References Phytase nP (%) AME Improvement

(FTU/kg) – + (%)

Martin and Farrel (1994) 1000-500 0.56-0.71 11.7 12.3 4.7

12.4 12.8 4.7
Selle et al. (1999) 600 0.50 12.6 12.9 2.6

600 0.39 12.4 12.9 4.0

Ravindran et al. (2000) 400 0.23 13.1 13.8 5.3

800 0.23 13.1 13.3 1.5

Ravindran et al. (2000) 400 0.45 12.6 13.1 3.9

800 0.45 12.6 13.4 6.3

Ravindran et al. (2001) 500 0.45 14.2 14.6 2.8

750 0.45 14.2 14.7 3.5

Selle et al. (2001) 600 0.25 14.2 14.1 -

nP=non-phytate P

62
non-phytate P) improved apparent faecal availability of Ca by 12% compared to an
adequate-P diet (0.45% non-phytate P). Sebastian et al. (1996) reported that the
addition of phytase to low-P diets significantly increased Ca retention by 12.2% in
male broilers but not in females. Qian et al. (1996, 1997) reported that phytase
supplementation improved Ca retention at various levels of non-phytate P and at
varying dietary Ca to total P ratios. Calcium retention increased linearly as the amount
of supplemental phytase increased, and decreased as the Ca to total P ratios became
wider.
2.6. Optimization of nutritional matrix in broilers fed corn-soy diets
Nutrient composition of different feed ingredients differs among different
geoclimatic regions. Such a composition, known as nutrient matrix of that particular
nutrient, would help better diet formulation to suit the bird’s needs. It is also essential
to have a ‘nutrient matrix of enzyme’ which would give us the level of improvement
in availability of different nutrients brought in by enzyme supplementation. This
would help in more accurate formulation of diets than the vague system of top-
dressing of enzymes, being followed in most parts of the country. More and more
enzyme manufacturers, therefore, are recommending their own ‘nutrient matrices’ for
their enzyme brands, developed after several biological experiments. Using such
nutrient matrix, one can reformulate diet reducing the levels of different nutrients as
per the level of enzyme inclusion for better performance and economy.