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7

RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE

The review of literature pertaining to the present study entitled

“Isolation and characterization of bacterial phytase and influence of

its feed supplementation in broilers” is discussed under the following

headings:

2.1 Poultry industry

2.2 Phytic acid chemistry

2.3 Enzymes in animal feed industry

2.4 Enzymology of phytase

2.5 Organic acids and their role in animal nutrition

2.6 Phytase and citric acid supplementation in poultry diet

2.1 POULTRY INDUSTRY

Livestock sector plays an important role in socio economic development

of rural households in India. In 2006, the livestock sector has grown at an

annual rate of 5.6%, which was higher than the growth of 3.3% agricultural

sector (Ali, 2007). Compared with the rest of the livestock sector, the poultry

industry in India is more scientific and well organized and progressing towards

modernization (Balakrishnan, 2002). The broiler chicken industry has now

occupied the second place in volume in the world just after pork (Yang and

Jiang, 2005). India is now the world's fourth largest egg producer and the fifth

largest producer of broilers (Saran et al., 2005). The layer industry is growing at

5 to 7% and the broiler industry at 10 to 12% per annum in India. The poultry

industry is contributing about Rs 350 billion to the national Gross Domestic

Product (GDP). In 2006, the poultry industry had the strength of 215 million

2

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layers and 1600 million broilers and by 2010 the growth rate was expected to

increase to 245 million layers and 2500 million broilers. According to the

Ministry of Food Processing Industry, in India duing 2007, the poultry industry

was estimated at over Rs 30,000 crore and is expected to grow over Rs 60,000

crore by 2010. However, the projected growth of industry depends to a great

extent on the availability of feed ingredients to meet the requirement of nearly

20 million tonnes of feed by 2010 (Sharma et al., 2006). Figure 1 presents the

projected poultry meat and egg consumption in India.

FIGURE 1

THE PROJECTED POULTRY MEAT AND EGG CONSUMPTION IN INDIA

(Mohanty and Rajendran, 2003)

Feed, is the largest cost in broiler and egg production, constituting 70 %

of the total. The main feed ingredients are maize, soy, rice bran and groundnut

cake. Maize and soy are the most widely used. Thus, the price movements of

these two feed items will have a direct effect on the prices of eggs and broilers.

Moreover, the future growth of the poultry industry will depend on the

availability and price of maize and soy. The industry has projected that demand

for maize in 2010 will be 16.65 million tonnes and foresees a major shortfall in

Millions

Million Kilograms

Poultry meat Egg

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maize production. The poultry sector consumes about 50% of the total maize

production followed by human consumption, other livestock, starch and

breweries. The situation is the same in case of soy as well (Metha et al., 2005).

2.2 PHYTIC ACID CHEMISTRY

Feeds of intensively reared poultry typically contain a high proportion of

cereals, grain legumes and oilseed meals. These feed ingredients contain

variable concentrations of phosphorus which ranges from around 19 g/kg in

extracted rice bran to less than 1g/kg in some tubers. However, approximately

two-thirds of the phosphorus is present as phytate phosphorus. Phytate is

ubiquitous in many types of plant material (Cowieson et al., 2006a). The use of

phytate phosphorus by single stomached animals has been reported to vary

from less than 10 to over 50% (Cowieson et al., 2006b). Phytic acid content of

different plant materials is summarized in Table 1.

2.2.1 Terminology related to phytic acid and phosphorus

The compound phytic acid can be commonly called as myoinositol

hexaphosphoric acid or scientifically 1, 2, 3, 4, 5, 6 hexakis (dihydrogen

phosphate) myoinositiol. In plants, phytic acid exists in its anionic form, phytate.

In mature seeds, phytic acid is found as a complex salt of calcium, magnesium

and potassium and in some cases it is bound to proteins and starches.

This complexed or chelated molecule of phytic acid is known as phytin

(Bohn et al., 2008).

The terms used for the different forms of phosphorus are as follows:

Total phosphorus is generally referred to any and all forms of phosphorus in the

diet. Available phosphorus (aP) refers to the phosphorus that is absorbed from

the diet by the animal (i.e., feed phosphorus minus phosphorus in the distal

ileum). Retained phosphorus refers to the phosphorus that stays in the body

(i.e., feed phosphorus minus excreta phosphorus).

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TABLE 1

PHYTIC ACID CONTENT IN DIFFERENT PLANT MATERIALS

Food Phytate (mg/g dry matter)

Food Phytate (mg/g dry matter)

Cereal based

French bread 0.3 - 0.4 Oat bran 12.4 - 29.6

Mixed-flour bread (70%wheat,30%rye)

0.2 - 0.7 Oat flakes 8.2 - 10.3

Mixed-flour bread (70%rye,30%wheat)

0 - 0.3 Oat porridge 7.7 - 10.6

Sourdough rye bread 0 - 0.3 Pasta 2.2 - 8.6

Wholewheat bread 4.3 - 6.8 Maize 11.5 - 14.2

Whole rye bread 2.5 - 4.8 Sorghum 5.6 - 9.8

Unleavened wheat bread

9.2 - 19.5 Rice (polished,cooked)

1.4 - 2.9

Corn bread 5.2 - 7.1 Wild rice (cooked) 16.4 - 20.1

Legume based

Chickpea 4.9 - 6.1 Navy beans (cooked)

7.4 - 10.6

Cowpea 5.8 - 10.3 Soybeans 9.9 - 14.9

Black beans 8.5 - 11.3 Tempeh 9.1 - 10.3

White beans 9.1 - 10.9 Tofu 8.2 - 9.3

Lima beans 6.2 - 9.8 Lentils (cooked) 6.5 - 9.3

Faba beans 10.1 - 13.7 Green peas (cooked)

5.7 - 7.8

Kidney beans

cooked

8.9 - 11.2 Peanuts 16.5 -19.1

Others

Sesame seeds (toasted)

43.2 - 55.1 Soy protein concentrate

12.4 - 21.7

Soy protein isolate 4.3 - 11.7 Amaranth grain 12.6 - 14.3

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FIG 2. STRUCTURE OF PHYTIC ACID

Most of the phosphorus in plant ingredients of seed origin is present in

the phytin molecule and is referred to as phytin phosphorus (PP). Any

phosphorus that is not bound to the phytin molecule is referred to as nonphytin

phosphorus (nPP). This nPP can be chemically determined by subtracting

analyzed phytin phosphorus from analyzed phosphorus. A key difference

between aP and nPP is that the term aP includes all absorbed forms of

phosphorus and will include inorganic P (Pi) and organic P (including

phytin phosphorus), whereas nPP excludes any PP available to the animal

(Angel et al.,2002).

2.2.2 Phytic acid structure

Phytic acid is the

hexaphosphoric ester of the

hexahydric cyclic alcohol

myoinositol (Kumar et al.,

2010). Myoinositol (1,2,3,4,

5,6) hexakisphosphate has six

groups of phosphates attached

to the inositol ring. Figure 2

gives the structure of phytic acid and table 2 gives the nomenclature and

structure of different myoinositol phosphate. Using the prefix “hexakis” instead

of “hexa” indicates that the phosphates are not internally connected and the

compound is consequently a polydentate ligand, which is a chelator that can

bind to more than one coordnation site of the metal atom. Each of the

phosphate groups is esterified to the inositol ring and together they can bind

upto 12 protons in total (Bohn et al., 2008). The acidity of the protons varies

from very strong acids to very weak (pKa up to 9.4) although ionic strength of

the solution and temperature influence these values (Torres et al., 2005).

Although various structural formulae for phytic acid have been proposed,

the two which have attracted the most attention are those of Neuberg and

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Anderson. The Neuberg structure implies 18 titratable hydrogen ions

whereas that of Anderson was 12. Evidences based on nuclear magnetic

resonance data and X-ray crystallography had established Anderson’s

structure (Evans et al., 1982).

TABLE 2

NOMENCLATURE AND STRUCTURE OF DIFFERENT MYOINOSITOL PHOSPHATES

Derivatives Common name Structure

Ins Myoinositol

InsP1(IP1) Myoinositol monokisphosphate

Ins(3)P1

InsP2(IP2) Myoinositol dikisphosphate

Ins(3,4)P2 Ins(3,6)P2

InsP3(IP3) Myoinositol trikisphosphate

Ins(3,4,6)P3 Ins(1,3,4)P3 Ins(1,4,5)P3

InsP4(IP4) Myoinositol tetrakisphosphate

Ins(1,3,4,6)P4 Ins(1,3,45)P4 Ins(1,4,5,6)P4

Ins(3,4,5,6)P4

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Derivatives Common name Structure

InsP5(IP5) Myoinositol pentakisphosphate

Ins(1,3,4,5,6)P5 Ins(1,2,3,4,5)P5 Ins(1,2,4,5,6)P5

Ins(1,2,3,4,6)P5

InsP6(IP6) Myoinositol hexakisphosphate

Ins(1,2,3,4,5,6)P6

2.2.3 Formation of phytic acid in plants

Normally the salts of phytic acid are found in plant seeds, animals and

soil but the acid originates from natural mineral sources containing phosphorus

or from fertilizers (Marchner, 1997). As phosphorus containing fertilizers are

applied to the soil, plant roots pick up the phosphate at a physiological pH

mainly as PO4-3 which remains as inorganic phosphorus (P) and is esterified

through the hydroxyl group to the carbon chain (C-O-P) as a single phosphate

ester or attached to another phosphate by an energy rich pyrophosphate bond.

The rate of exchange between phosphorus and organic phosphorus in the

ester and the pyrophosphate bond is very high and this leads to plant roots

incorporating phosphorus into the organic phosphorus within a few minutes but

this is released almost immediately again into the xylem (Pelig-Ba, 2009).

Hence the proportion of total phosphorus found as phytate phosphorus

increases with the dose of phosphorus supplied to the plant. When a plant

receives a higher dose of phosphorus than it requires, surplus phosphorus

seems to be stored in the form of phytic acid. Figure 3 gives the biochemical

pathway of phytic acid formation from glucose 6 phosphate.

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FIGURE 3

BIOCHEMICAL PATHWAYS IN THE FORMATION OF PHYTIC ACID FROM GLUCOSE 6 PHOSPHATE

(Raboy, 2003)

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2.2.4 Localization of phytic acid in plants and animals

Phytin is found primarily in seeds, with its location within the seed

differing among plants (Angel et al., 2002). Ninety percent of the phytin in corn

was reported to be found in the germ portion of the kernel, whereas in wheat,

barley and rice the majority of phytate was found to be accumulated in the

aleurone cells and only minor amounts in the embryo (Bohn et al., 2008).

Phytate content was found to be much higher in the bran than in the whole

grain (Guttieri et al., 2003; Steiner et al., 2007) but also within the bran fractions

differences were noticed.

In most oilseeds and grain legumes, phytin is associated with protein

and concentrated within subcellular inclusions called globoids that are

distributed throughout the kernel; however, in soybean seeds, there appeared

to be no specific location for phytin (Angel et al., 2002). Globoids are

compartmentalised inside protein storage vacuoles in the seeds. The size of

the phytate globoids was found to depend on the amount of phytate in the

grain. In wildtype wheat, globoids upto 4 micrometer in diameter have been

detected (Antoine et al., 2004), whereas a wheat mutant with lower phytic acid

had smaller globoids, organised in clusters (Joyce et al., 2005). Location of

phytin within the seed and its chemical associations with other nutrients

seemed to influence its availability (Bohn et al., 2008).

Although most phytic acid is located in the seeds, there are plants in

which it appears to be distributed throughout. Small amounts have been found

in the aerial parts of several grass species and underground parts of carrots,

potatoes and artichokes (Abernethy et al., 1973).

Presence of phytate in animal tissue is found in a lesser extent.

Grases et al. (2001) have demonstrated using gas chromotography (GC) mass

detection methodology that highest phytate accumulation was found in brain of

rats when they were fed on diets with different phytate contents.

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2.2.5 Role of phytic acid in plants and animals

The salt form, phytate, is an anhydrous storage form of phosphate

accounting for more than 80% of the total phosphorus in cereals and legumes.

Phytic acid is also a storage form of myoinositol – an important growth factor.

The lower inositol phosphates (IP1-4) and myoinositol have been recognized as

beneficial through different biological roles (Reale et al., 2007).

Phytic acid is utilized during seed germination. In seeds, the role of

phytin is as follows: 1) a phosphorus reserve, 2) an energy store, 3) a

competitor for adenosine triphosphate during rapid biosynthesis of phytin

near seed maturity when seed metabolism is inhibited and dormancy is

induced, 4) an immobilizer of divalent cations required for the control of

cellular processes and 5) a regulator of readily available seed phosphate level

(Angel et al., 2002; Raboy, 2003). Lower inositol phosphates were found to be

involved in stress responses, membrane biogenesis and intracellular signalling

(Storcksdieck et al., 2007).

In mammalian organisms, phytic acid has been implicated in starch

digestibility and blood glucose response (Lee et al., 2006), in the prevention of

dystropic calcifications in soft tissues (Grases et al., 2004), kidney stone

formation (Selvam, 2002), in the lowering of cholesterol and triglycerides

(Onomi et al., 2004) and apparently it has been tested in toothpaste as a tool

for preventing plaque formation (Vasca et al., 2002). Numerous studies in the

medical literature have reported phytate as a broad spectrum antineoplastic agent

(Vucenik and Shamsuddin, 2003).

2.2.6 Antinutritional properties of phytic acid

Due to its chemical structure, phytic acid is a very stable molecule. It

differs from other organophosphate molecules in having a high phosphate

content, which results in a high negative charge over a wide pH range and

therefore its presence in the diet has a negative impact on the bioavailability of

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divalent and trivalent mineral ions such as Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+ and

Cu2+ (Fredlund et al., 2006). Besides, phytate has also been reported to form

complexes with proteins at both low and high pH values. These complex formations

alter the protein structure, which might result in decreased protein solubility,

enzymatic activity and proteolytic digestibility (Kumar et al., 2010). Thus, phytic acid

is an antinutritive component in plant derived food and feed.

Furthermore, phosphorus in the form of phytic acid is largely unavailable

as a nutritional factor to monogastric animals because insufficient degradation

capabilities in the gastrointestinal tract prevent the phosphorus from being

biologically available. The excess of phosphorus bound in phytic acid is then

excreted through the faeces and spread as manure into the soil. The potential

eutrophication of fresh water streams, lakes and near coastal areas can then

cause cyanobacterial blooms, hypoxia and death of aquatic animals and

production of nitrous oxide, a potential green house gas (Vats et al., 2005).

The antinutritional effect of phytic acid causes deficiency related

diseases in monogastrics which might lead to poor quality egg and meat in

case of poultry. Phytic acid is also partially attributed to the wide spreading

human nutritional deficiencies of calcium, iron and zinc in developing countries

where the staple foods are of plant origin (Manary et al., 2002).

Phytate-metal ion complexes

The presence of phytate in the diet has a negative effect on

mineral uptake (Konietzny and Greiner, 2003; Lopez et al., 2002).

Binding assays using phytic acid show the order of binding ions as

Cu2+>Zn2+>Ni2+>Co2+>Mn2+>Fe3+>Ca2+ (Vohra et al., 1965). Despite these

results, the composition of minerals in phytic acid stores does not necessarily

follow these orders of affinity. Recent findings show that phytic acid is stored

in vivo in complexes not only with these minerals, but to a much larger extent

with magnesium, calcium and potassium (Bohn et al., 2007).

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In presence of excess phytic acid, formation of soluble complexes

between phytic acid and a metal ion displaying 1:1 stoichiometries

predominates. However, when metal ions are in excess, an insoluble solid

called phytate is formed (Torres et al., 2005) (Figure 4). In human studies,

phytic acid has been reported to inhibit absorption of iron, zinc, calcium,

magnesium and manganese but surprisingly not copper (Bohn et al., 2004;

Egli et al., 2004; Phillippy, 2006). Iron and zinc uptake have both been shown

to be inhibited when the phytic acid: metal ratio was increased above

10:1 (Gharib et al., 2006; Glahn et al., 2002). Studies on the effects of phytate on

dietary manganese, copper and magnesium are limited (Lopez et al., 2002).

Removal or degradation of phytic acid would therefore increase the

bioavailability of many cations and improve nutritional value of the meal

(Bohn et al., 2008).

FIGURE 4

STRUCTURE OF PHYTATE - METAL COMPLEX

Phytate-protein interactions

Phytate forms a strong complex with some proteins and resists their

proteolysis (Kumar et al., 2010). The interaction between phytate and protein is

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largely dependent on pH (Adeola and Sands, 2003). At a pH value lower than

the isoelectric point of proteins, phosphoric acid groups of phytate bind with the

cationic group of basic amino acid and form binary protein-phytate complexes.

They are insoluble complexes that dissolve only below pH 3.5. Such complex

formations might affect the protein structures that can hamper enzymatic

activity, protein stability and protein digestibility (Kumar et al., 2010). In vitro

studies showed that the extent of phytate-protein interaction is governed by

various factors, including pH, the source and solubility of protein and dietary

levels of calcium and magnesium (Kemme et al., 1999). If the steric conditions

are satisfactory, one phytate anion can interact with two charged groups of

protein. Naturally, the protein molecule can bind more phytate anions at the

same time, depending on the number of positively charged groups and

conformational conditions (Hidvegi and Lasztity, 2002).

It was also shown that phytate-protein complexes are less likely to be

digested by proteolytic enzymes (Cowieson et al., 2006b) and even digestive

enzymes such as pepsin, trypsin, chymotrypsin, lipase and amylase are

inhibited by phytate. This inhibition might be due to the non specific nature of

phytate- protein interactions and the chelation of calcium ions, which are

essential for the activity of trypsin and alpha amylase. The reduction in the

protease activity might also be partially responsible for poor protein digestibility

(Kumar et al., 2010).

Phytate-lipid interactions

Phytate forms lipophytins (complexes with lipid and its derivatives) along

with other nutrients (Vohra and Satyanarayana, 2003). Lipid and calcium

phytate might be involved in the formation of metallic soaps in gut lumen of

poultry, which is a major restraint for energy utilization derived from lipid

sources (Leeson, 1993). Young chicks, when fed with diets supplemented with

fat and phytate, exhibited hampered phytate phosphorus utilization and a large

percentage of fat was excreted as soap fatty acids (Matyka et al., 1990).

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Phytate-carbhohydrate interactions

Phytate intake was found to reduce the blood glucose response

(Lee et al., 2006). Phytate might have formed complexes with carbohydrates of

feedstuffs thereby reducing their solubility, adversely affecting the digestibility

and absorption of glucose. Phytate might bind with starch either directly via

hydrogen bonds or indirectly via proteins associated with starch (Rickard and

Thompson, 1997). It was suggested that ternary complexes of protein, phytic

acid and carbohydrate formed subsequently might affect the digestion rate of

starch (Hidvegi and Lasztity, 2002).

It is evident that the effects of phytic acid are attributed to its ability to form

complexes with positively charged food components, such as proteins,

carbohydrates, minerals and trace elements.

2.2.7 Toxicology of phytic acid

The presence of biologically active components such as phytates and

phenolic compounds were found to have adverse effects on intrinsic properties

of proteins. Phytic acid represents a complex class of naturally occurring

organic form of phosphorus compounds that can significantly influence the

functional and nutritional properties of foods (Sabah and Murwa, 2010).

Toxicological aspects of phytic acid were extensively studied on some of the

animal systems that included human.

Impaired magnesium bioavailability due to 15 g phytic acid/kg diet was

found to accompany by an increase in hepatic thiobarbituric acid reactive

substances and protein carbonyls as well as by a moderate decline in liver

reduced glutathione levels. The liver homogenates of rats receiving the diets

with 7.5 and 15 g phytic acid/kg respectively were much more susceptible to

iron induced lipid peroxidation than those of the controls (Rimbach and Pallauf,

1999). However, phytic acid did not seem to have any scavenging effect on

superoxide radicals generated in the xanthine/xanthine oxidase reaction.

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At marginal dietary iron supply, phytic acid supplementation reduced

apparent iron absorption, thereby decreasing liver iron concentration. Neither

phytic acid nor iron had any significant effect on liver oxidant or antioxidant

status in vivo in growing rats (Rimbach and Pallauf, 1998).

Lees and Leong (1996) have reported phytic acid toxicity to neuronal

perikornia when injected into the rat hippocampus. Its potency as a toxin was

approximately equal to that of the excitotoxin quinolinate. It was concluded by

their study that abnormal metabolism of phytic acid might possibly contribute to

neuronal death in neurodegenerative diseases. Carcinogenecity of phytic acid

has been studied and Hiasa et al. (1992) have shown necrosis and calcification

of renal papillae in Fischer 344 rats when treated with phytic acid. The rate of

colonic cancer could arise from many fibre rich foods which might contain high

phytate (Vucenik and Shamsuddin, 2003).

2.3 ENZYMES IN ANIMAL FEED INDUSTRY

Consistent increase in the price of feed ingredients has been a major

constraint in most of the developing countries. As a result, cheaper and

nonconventional feed ingredients have to be used which contain higher

percentage of nonstarch polysaccharides (soluble and insoluble/crude fibre)

along with starch (Khattak et al., 2006). In addition, consumer awareness and

new legislation require that any increase in animal production cannot be

achieved via growth promoting drugs or other chemical substances.

Consumers and industrialists are looking more closely than ever before into

how compound animal feeds are produced, how the animals are reared, the

current feeding practices and the end result of the systems of animal

husbandry to the environment (Godfrey and West, 1996). Among all, the role of

feed and nutrition for livestock production has gained attention as it can affect

the animal directly which in turn can affect the human health and the

environment (Sapkota et al., 2007).

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2.3.1 Different enzymes used in feed

The major types of enzymes used in animal feeds are categorized by

their substrate specificity. All enzyme preparations recommended for use to

improve dietary nutrient utilization in animals are found to be hydrolases

(Ferket and Santos, 2006). Commercial enzyme products are typically a blend

of several different enzymes that are effective on a wide variety of substrates

that comprise the feed. The enzymes with proven efficacies for animal

husbandry include xylanase, arabinoxylanase, β-glucanase, cellulase, phytase

(Choct and Kocher, 2000), proteases (Khattak et al., 2006) and phospholipase

(Santos et al., 2004).

For most of the enzymatic processes, microbial enzymes are preferred,

primarily because they are easier and cheaper to obtain. Microorganisms

secrete a wide variety of proteolytic enzymes. These enzymes are of

central importance and are among the most important hydrolytic enzymes

(Haq and Mukhtar, 2006). The use of enzymes impart less chemical

load on the environment, higher efficiency and the ability to dilute multiple

downstream transformation attempts while maintaining product yield and

recovery (Chandel et al., 2008).

The first commercial success was the addition of β-glucanase into

barley based feed diets. Barley contains β-glucan, which causes high viscosity

in the chicken gut. The net effect of enzyme usage in feed was reported to be

increased animal weight gain with the same amount of barley resulting in

increased feed conversion ratio. Later enzymes were also tested in wheat

based diets. Silva and Smithard (2002) studied the effect of xylanase on rye

based diets and observed that the reduction in small intestinal viscosity by the

enzyme improved nutrient digestion. Xylanases are nowadays routinely used in

feed formulations (Ferket and Santos, 2006).

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Another type of important feed enzyme is phytase. It is a

phosphoesterase which liberates phosphate from phytic acid which is a

common compound in plant based feed materials. The net effect is reduced

phosphorus in faeces resulting in reduced environmental pollution. The use of

phytase reduces the need to add phosphorus to the feed diet. Supplementation

of microbial phytase was observed to reduce the need for mineral

supplementation by increasing the availability of cations (phosphorus, calcium,

zinc and copper) bound to phytic acid (Odetallah, 2000).

The action of cellulase is complex because cellulose is generally

associated with other polymers, such as lignin and pentosans. Recently,

protease on corn soybean meal based diets has received considerable

attention. Supplementation of poultry diets with enzyme mixtures that include

proteases and amylases have produced significant improvements in growth

performance (Greenwood et al., 2002; Burrows et al., 2002).

Usually a feed enzyme preparation is a multienzyme cocktail containing

glucanases, xylanases, proteinases and amylases (Ferket and Santos, 2006).

They are normally incorporated into the grains before pelletization and the

feed briefly reaches processing temperatures of 85 - 90°C. Therefore the feed

enzymes need to be thermo tolerant (Rao et al., 2009).

2.4 ENZYMOLOGY OF PHYTASE

Lott et al. (2000) have estimated that nearly 35 million metric tons of

phytic acid, containing 9.9 million metric tons of phosphate, is combined

with about 12.5 and 3.9 million metric tons of potassium and magnesium

respectively, to form each year over 51 million metric tons of phytate. The

amount of phosphorus in this phytate was found to be equal to nearly 65% of

the elemental phosphorus sold worldwide for use in mineral fertilizers. Dry

cereal grains account for 69% of the total crop seed/fruit production but account

for 77% of the total phytic acid stored each year.

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Decrease in phytic acid was found to be very advantageous, due to its

influence on nutrition and therefore interest has grown to reduce its

antinutritional effects (Abdelrahaman et al., 2005). Also the mineral binding

capacity was found to decrease when the phosphate groups were removed

and this might reduce the adverse effects of phytate on mineral bioavailability

(Kim et al., 2008).

It is estimated that 10 kg of dicalcium phosphate can be replaced by

250 g of phytase enzyme (Gulati et al., 2007). In India, the use of phytase in

monogastric diets was estimated to be approximately 500 tons/year as reported

by Compound Livestock Feed Manufacturers Association (CLFMA, 2007).

But the phytase market in China was reported to be 5500 tons/year (Xu, 2006).

In biological system, hydrolysis of phytic acid to myoinositol and

inorganic phosphate is an important reaction for energy metabolism, metabolic

regulation and signal transduction pathways. The reaction was primarily

catalysed by phytases (myoinositol hexakisphosphate phosphohydrolase).

They are histidine acid phosphatases (HAPs), a subclass of phosphatases,

which catalyze the hydrolysis of phosphate moieties from phytic acid, thereby,

resulting in the loss of ability of phytic acid to chelate metal ions (Vats and

Banerjee, 2004).

2.4.1 Classification of phytase

The International Union of Pure and Applied Chemistry and the

International Union of Biochemistry and Molecular Biology (IUPAC-IUBMB)

have recognized three classes of phytase enzymes, 3-phytases, 5-phytases

and 6-phytases, which initiate the dephosphorylation of phytic acid at different

positions on the inositol ring and produce different isomers of the lower inositol

phosphates (Bohn et al., 2008).

Within each class of phytase, structural differences can be found and not

all enzymes within a certain class hydrolyze phosphate from phytic acid

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through the same mechanism. All phytases have pronounced stereo specificity

and a strong preference for equatorial phosphate groups over axial groups

(Lei and Porres, 2003). Figure 5 shows the end products of phytate hydrolysis

mediated by phytase enzyme from different origins.

The largest group of phytases belong to the 3-phytases (EC 3.1.3.8),

which in general are found in fungi and bacteria and initiate the hydrolysis of

phytate at the third phosphate group (Sajidan et al., 2004). Structurally,

most of the 3-phytases show homology to betapropeller phosphatase (BPP) or

histidine acid phosphatases (HAP). BPPs are tightly bound to three calcium

ions and two phosphate groups before hydrolysis can occur. The end

product has been suggested to be inositol triphosphate either Ins(1,3,5)P3 or

Ins(2,4,6)P3 (Kerovuo et al., 2000; Shin et al., 2001), but Oh et al. (2006)

reported Ins(2,4,6)P3 to be the sole end product, confirming the equatorial

preference of most phytases.

FIGURE 5

THE END PRODUCTS RESULTING FROM PHYTATE HYDROLYSIS MEDIATED BY DIFFERENT PHYTASES

Bacillus phytases

Fungal phytases

Plant phytases

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HAPs can initiate hydrolysis of phytic acid on either the C3 or the C6

position of the inositol ring and produce myoinositol monophosphate (in

particular Ins(2)P due to its axial position) as the final product (Mullaney

and Ullah, 2003; Greiner and Carlsson, 2006; Oh et al., 2006). Most bacterial,

fungal and plant phytases belong to the HAPs. Within this structural

classification, there are two phytase subgroups: some show broad

substrate specificity but low specific activity for phytic acid, whereas others

have narrow substrate specificities but high specific activity for phytic acid

(Bohn et al., 2008).

6-phytases (EC 3.1.3.26) initiate the hydrolysis of phytate at the sixth

phosphate group (Greiner et al., 1993; Kaay and Haastert, 1995). Several

structurally different phytases namely, the purple acid phosphatase (PAP), the

ADP phosphoglycerate phosphatase (related to EC 3.1.3.28) and an HAP-class

are found in this group (Bohn et al., 2008). Purple acid phosphatase is found as

a homodimeric glycoprotein in mainly plant species, with a Fe(III)-Zn(II) active

site (Dionisio et al., 2007).

The 3-phytases do not always completely dephosphorylate IP6, whereas

the 6-phytases do (Angel et al., 2002). According to the conserved domain

database (CDD) of the National Center for Biotechnologyl Information (NCBI),

the conserved domains in acid phosphatases, phytases and purple acid

phosphatases are designated as pfam00328, pfam02333 and pfam02227

respectively. 5-phytases (EC 3.1.3.72) from Medicago sativa, Phaseolus

vulgaris, and Pisum sativum which were found to initiate phytate hydrolysis at

the fifth phosphate group (Rao et al., 2009).

2.4.2 Occurrence of phytase

Natural degradation of phytic acid was observed to be almost

impossible and chemical hydrolysis in the laboratory was found to be very slow

(Turner et al., 2002). However, the enzyme phytase is found wide spread in

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nature (including animals, plants and microbes) which can rapidly breakdown

phytate (Mullaney and Ullah, 2003).

Plant and animal phytase

Phytase occurs widely in the plant kingdom. Phytase has been isolated

and characterized from cereals such as triticale, wheat, maize, barley and rice

and from beans such as navy beans, mung beans, dwarf beans and California

small white beans. Phytase activity has also been detected in white mustard,

potato, radish, lettuce, spinach, grass and lily pollen (Dvorakova, 1998).

Different phytases were identified in negligible quantities in the mucosal

extracts of small intestine of rats, chickens, humans, rabbits and guinea pigs

(Marounek et al., 2003).

Microbial phytase

Researches have shown that microbial phytases are most promising

for biotechnological applications (Vohra and Satyanarayana, 2003). Although

phytases from several species of bacteria, yeast and fungi have been

characterized and commercial production was mostly focused on the soil

fungus Aspergillus. However, due to some properties such as substrate

specificity, resistance to proteolysis and catalytic efficiency bacterial phytases

have proved to be real alternative to the fungal enzymes (Konietzny and

Greiner, 2004).

Phytases have been detected in various bacteria, such as

Pseudomonas sp. (Richardson and Hadobas, 1997), Bacillus sp. (Kerovuo

et al., 1998; Choi et al., 2001), Raoultella sp. (Greiner et al., 1997; Sajidan

et al., 2004), Escherichia coli (Greiner et al., 1993), Citrobacter braakii

(Kim et al., 2003), Enterobacter (Yoon et al., 1996), Lac. Sanfranciscensis

(Angelis et al., 2003) and anaerobic rumen bacteria, particularly in

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S. ruminantium, M. elsdenii, Prevotella sp., M.multiacidus (Yanke et al., 1998)

and M. jalaludinii (Lan et al., 2002a).

Generally, the phytases produced by fungi are extracellular, whereas the

enzymes from bacteria are mostly cell associated. The only bacteria showing

extracellular phytase activity are those of the genera Bacillus (Kim et al., 1998;

Choi et al., 2001) and Enterobacter (Yoon et al., 1996). The phytases of E. coli

have been reported to be periplasmatic enzymes (Greiner et al., 1993) and

phytase activity in S. ruminantium and M. multiacidus were found to be

associated with the outer membrane (D’Silva et al., 2000).

2.4.4 Current and potential applications of phytase

In animal feed

Phytase has been mainly, if not solely, used as a feed supplement in

diets largely for swine and poultry and to some extent for fish. Numerous

laboratory experiments and field trials have shown that 500 to 1000 units of

phytase can replace approximately 1 g inorganic phosphorus supplementation

and reduce total phosphorus excretion by 30 - 50% (Kemme et al., 1997;

Lei et al., 1993a; Liu et al., 1997; Yi et al., 1996a). The benefits of phytase

are two fold: saving the expensive and nonrenewable inorganic phosphorus

resource by reducing the need for its inclusion in animal diets and

protecting the environment from pollution of excessive manure phosphorus

runoff. Supplemental organic acids such as citric acid or lactic acid were

found to enhance phytase efficacy (Han et al., 1998; Jongbloed et al., 2000;

Maenz et al., 1999).

In human nutrition

Processing and manufacturing of human food is also a possible field of

application of phytase. No phytase product for a relevant food application is

reported to be on the market till now. Research in this field focuses on better

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mineral absorption or technical improvement of food processing (Haefner et al.,

2005). Some food processing methods are shown to reduce or remove

considerable amounts of phytate in legumes (Awada et al., 2005; Soetan

and Oyewole, 2009) but use of phytase could reduce the phytic acid content in

food products more efficiently. Phytase was reported to fully degrade phytic

acid during the manufacture of roller dried complementary foods based on

flours from rice, wheat, maize, oat, sorghum and a wheat soy flour blend

(Hurrell et al., 2003). Haros et al. (2001) investigated the possible use of

phytase in the process of bread making. The main achievement of this use was

the shortened fermentation period without affecting the bread dough pH. An

increase in bread volume and an improvement in crumb texture were also

observed.

In the synthesis of lower inositol

Lower phosphoric esters of myoinositol (mono, bis, tris, and

tetrakisphosphates) play a crucial role in transmembrane signaling processes

and in calcium mobilization from intracellular store in animal as well as in plant

tissues (Dasgupta et al., 1996; Krystofova et al., 1994). The use of phytase has

been shown to be very effective in producing different inositol phosphate

species. Phytase isolated from A. niger was shown to efficiently hydrolyze IP6

to all lower phosphorylated derivatives from myoinositol pentakisphosphate

(IP5) to myoinositol dikisphosphate (IP2) depending on the amount of the

enzyme (Dvorakova et al., 2000).

2.5 ORGANIC ACIDS AND THEIR ROLE IN ANIMAL NUTRITION

Besides the nutritional value, high quality feed also has a positive effect

on the health status of animals. The most efficient way to keep compound feed

at a high hygienic status is the use of organic acids. Acids with an antifungal

activity provide an efficient way to ensure the quality of stored cereals, other

feed raw materials and compound feed (Celik et al., 2003). Organic acids

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(C1-C7) are widely distributed in nature as normal constituents of plants or

animal tissues. They are sometimes found as their sodium, potassium or

calcium salts or even as stronger double salts. Citric acid, fumaric acid,

propionic acid, lactic acid and formic acid are the commonly used acids in the

poultry diets.

2.6 PHYTASE AND CITRIC ACID SUPPLEMENTATION IN POULTRY DIET

2.6.1 Growth performance

Studies with chicks and turkeys have indicated a positive response in

growth performance from the addition of various organic acids to diets (Celik

et al., 2003; Owen et al., 2008). Gunes et al. (2001) and Adil et al. (2010) have

reported that the body weight gain was significantly improved by dietary

supplementation of organic acids when compared with the control group.

Yan et al. (2000) reported an increase in body weight at 21 day

in broilers with the addition of phytase. Many studies (Ahmad et al., 2000;

Pintar et al., 2004 and Bozkurt et al., 2006) have reported an increase in weight

gain with increasing concentration of dietary phytase in soybean meal based

diets. However, Pizzolante et al. (2002) have found no effect on weight gain of

broilers due to phytase addition. These differences among studies might have

resulted from the amount of available phosphorus used and amount of phytase

added in diets (Bingol et al., 2009).

Bingol et al. (2009) have observed that broilers fed negative control diet

had significantly lower feed efficiency compared with other treatment groups

that were supplemented with phytase. Bozkurt et al. (2006) noticed that feed

efficiency of broilers fed diet containing phytase were similar to those fed

control diet with adequate phosphate. Feed conversion ratio was found to be

better with phytase supplementation in broilers (Tejedor et al., 2001). Park et

al. (2009) and Sharafat et al. (2009) have reported better feed conversion ratio

when citric acid was added to the diet of layers. Omogbenigun et al. (2003)

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have noted a better feed conversion ratio in piglets when the diet was

supplemented with both phytase and organic acid.

The mortality rate observed during the study period was attributed to

phosphorus requirement. It was reported that not enough phosphorus was

available to the chicks (Sohail and Roland, 1999). Punna and Roland (1999)

have reported that phosphorus requirement of chicks varied with the age, size

and strain of birds. It was reported that phytase supplementation to moderate

and low nonphytate phosphorus level increased livability percentage

(Scheideler and Ferket, 2000).

The improvement in the performance of broilers fed with phytase

supplementation can be explained by the improved utilization of energy,

protein, amino acids, as well as macro and microminerals (Santos et al., 2008).

2.6.2 Mineral retention

Leeson and Summers (2001) stated that minerals are inorganic

materials occurring in nature in the form of salts or mixed with organic

compounds. The availability of minerals to animals and their metabolic

functions largely depend on the compound in which they are present.

Phosphorus, when combined with phytic acid is not available to monogastric

animals. Despite the adequacy of certain elements in the diet, deficiency

symptoms were observed at times.

Adding microbial phytase in combination with citric acid decreased the

phosphorus disappearance in the crop (a portion of the digestive system as

indicated in Figure 9) content compared to birds which were supplemented only

with phytase in laying hens (Ali et al., 2009). Viswanathan et al. (2007) have

also reported that 750 U of phytase plus 3% citric acid supplementation

improved the digestibility of phosphorus than control diet in pigs. Addition of

500 U microbial phytase/kg and 0.35% organic acid mixture composed mainly

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of citric acid improved phosphorus digestion and utilization, thereby leading to a

reduction in phosphorus excretion (Omogbenigun et al., 2003).

Viverous et al. (2002) had reported that compared to the normal

nonphytate phosphorus diet, the birds fed with low nonphytate phosphorus

diets without phytase had decreased calcium retentions at 3 (up to 30%) and 6

weeks of age (up to 25%). Phytase supplementation to the low nonphytate

phosphorus diets increased calcium retention at 3 weeks by 22% and 6 weeks

of age by 15%. Radcliffe et al. (1998) have reported that calcium digestibility

was improved by the addition of citric acid. Calcium retention was increased by

supplementation of both phytase and citric acid in broilers (Agustin et al.,

2003). The improvement in calcium availability has been expected because

phytase liberates calcium from the calcium-phytate complex and as the

availability of phosphorus increases, the availability of calcium also increases

(Ahmad et al., 2000).