Protease for use in poultry

INTRODUCTION
Most microorganisms and plants can biosynthesize all 20 standard amino acids, while animals
(including humans), must obtain some of the amino acids from the diet. Key enzymes in the animals’
body amino acid biosynthetic for specific amino acids are not present at all or not present in enough
quantity in animals. The amino acids that an organism cannot synthesize, or synthesized at an
insufficient quantity, are referred to as essential amino acids. If amino acids are present in the
environment, microorganisms can conserve energy by taking up the amino acids from their
surroundings and downregulating their biosynthetic pathways and thus they start requiring outside
sources of these amino acids.
In animals, amino acids are obtained through the consumption of foods containing protein. Ingested
proteins are broken down through digestion, which typically involves denaturation of the protein
through exposure to acid and hydrolysis by enzymes called proteases. Some ingested amino acids are
used for protein biosynthesis, while others are converted to glucose through gluconeogenesis, or fed
into the citric acid cycle. This use of protein as a fuel is particularly important under starvation
conditions since it allows body proteins - particularly those found in muscle - to be used to support life.
Amino acids are also important dietary sources of nitrogen.
(http://www.dsm.com/en_US/downloads/dnpna/Proteases_Potential_for_Use_in_Poultry_Nutrition.p
df)
Digestion is the process of breaking down large, complex molecules, as provided by the birds’ feed, into
smaller components that can be absorbed into the portal blood system. The process involves changes in
both physical and chemical structures of most dietary components. Poultry feeds consist of a complex
array of particles differing not only in chemical composition, but also in size, hardness, solubility and
ionic characteristics.
PROTEASE FOR USE IN POULTRY
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Under ideal conditions, this array of particles and chemicals with different characteristics degrade
slowly in a step-wise manner as feed passes from the mouth to the large intestine. Particle breakdown
is a constant process, although the gizzard provides the major site of this activity.
In 1877, Moritz Traube proposed that (i) protein - like materials catalyzed fermentation and other
chemical reactions and (ii) they were not destroyed by doing such things. This was the beginning of the
recognition of what we call enzymes today.
Enzymes are largely responsible for molecular degradation, although their pH greatly influences their
efficacy.
When digestion is reduced, there will be reduced bird growth and/or increased feed intake. Indigestion
may also cause problems with manure/litter management, because non-digested residues in the large
intestine often adsorb more water or produce feces that are more viscous.
Animal feed contains Cereals and Cakes.
Cell walls of cereals are primarily composed of carbohydrate complexes referred to as Non Starch
Polysaccharides (NSP).
ANFs present in these NSP (like ß-glucansand arabinoxylans) are non digestible and form high-molecular-weight
viscous aggregates in the gastrointestinal tract.
They
Affect the digestive enzymes.
Cause endogenous losses
Reduce the rate of passage.
Stimulates pathogenic microbial proliferation.
Protease can degrade the proteins.
ANF
PALM
The anti-nutritional factors of the palm kernel cake as discussed by Aletor (1999) are those chemical compounds
synthesized inherently or naturally by one organism, acting as stimulators or inhibitors; the factors are phytic
acid, tannic acid, oxalate contents and phytin phosphorus.
Anti-nutritional factors of Palm Kernel Cake samples
Anti-nutrients E1 E2 Mean SD CV (%)
Tannic acid (%) 0.35 0.44 0.40 0.064 16.0
Phytin Phosphorus (mg/g) 6.50 6.73 6.62 0.163 2.46
Phytic acid (mg/g) 23.07 23.90 23.49 0.587 2.50
Oxalate (mg/g) 5.04 5. 22 5. 13 1.273 24.18
E1 and E2 are duplicate determinations.
SD = Standard Deviation; CV = Coefficient of variation at P < 0.05 significant level.
SOY
Soyabean seeds have certain anti-nutritional factors like trypsin inhibitors, protein inhibitors, protease
inhibitors, phytohemagglutinin, saponins, goitrogen and estrogenic factors that can affect the production

performance of chickens. But they are all thermo labile and can be destroyed by roasting, heating or
autoclaving.
Sesame/Til
Sesame (sesamum indicum) meal contains 40% protein and 8% crude protein fiber. The protein is rich in
arginine, leucine and methionine but low in lysine. Since the sesame meal is deficient in lysine its combination
with Soyabean meal appears to be useful. Sesame meal has high calcium and phosphorus content but their
availability is low because of higher phytate content in the hulls of the seed. The phytic acid reduces the
availability of calcium, zinc, magnesium, copper etc. from the diet. Nevertheless, the meal can be included in
poultry diets up to 15% in combination with other lysine rich oil cakes.
Sunflower
Sunflower meal is generally not recommended be yond 20% in the diets may be due to high crude fiber content
in the meal and also due to the presence of a polyphenolic compound called chlorogenic acid which inhibits the
activity hydrolytic enzymes. To overcome this problem, supplementation of additional methionine and choline
in the diet is suggested.
Cotton seed
Quality of cotton seed meal is poor due to the presence of gossypol, phenol like compounds that are present in
the pigment gland of cotton seed. Gossypol inhibits the activity of digestive enzymes and reduces the
palatability of diet. Mechanical pressing of seed followed by solvent extraction reduce the gossypol content to
0.02-0.5% level. Dietary levels of gossypol up to 0.015% levels are believed to be safer in poultry. Detoxification
cotton seed meal with solvent mixture containing hexane, acetone and water were found to be useful after
initial cooking of the meal. Addition of ferrous Sulphate at the rate of 4 parts to one part of gossypol prevented
yolk discoloration of the eggs due to gossypol. Cotton seed has a desirable profile of amino acids except lysine
and the digestibility of cotton seed meal is poor due to higher crude fiber.
Caster seed
The protein content of caster (Ricinus communis) seed meal varies from 21 to 48% depending upon the extent of
decertification and oil extraction. It has an ideal amino acid profile with moderately high cystine, methionine
and isoleucine. But its antinutritional substances called the ricin, ricinine and an allergen restricts its use in
poultry even at low levels of inclusion. Processing of caster meal by heating at 80 c temperature in the presence
of GN ammonia or an acid or alkali reduces the toxic content substantially. Detoxified meal needs to be
effectively corrected for lysine.
G.I Tract Region Enzyme (or
secretion)
Substrate End Product pH
Mouth Saliva Lubricates and softens food
Crop Mucus Lubricates and softens food 4.5
Stomach (Gizzard
and proventiculus)
HCI Lowers stomach pH 2.5
Pepsin Protein Polypeptides

Duodenum Trypsin,
Chymotrypsin and
Elastases
Proteins, Peptones
and Peptides
Peptones,
Peptides and
amino acids
Carboxy-petidases Peptides Peptides and
amino acids
6
Collagenase Collagen Peptides to
6.8
Jejunum Peptidases Peptides Dipeptides and
amino acids
5.8 to
6.6
Polynucleotidase Nucleic acids Mono-
nucleotides
Posttranslational glycosylation has been reported to protect enzymes from deactivation caused by high
temperatures and proteinases
(Olsen and Thomsen, 1991).
All enzyme feed additives are considered either food additives or GRAS substances
The activity of enzymes retains more than 95% when stored at the temperature of 25 Deg C upto 3 months
PROTEINS:
Protein and amino acid availability are of greatest concern in animal and vegetable protein ingredients. Protein
content and availability from cereals and their by-products seem to be more consistent and little affected by
processing conditions.
Feedstuff C. Protein (%) Digestibility (%)
C. Protein Lys Met Cys
Vegetable sources (cereals)
Yellow maize 8 82 - 86 81 91 85
Wheat 12 78 - 82 81 87 87
Barley 10 70 -82 78 79 81
Sorghum 10 67 - 72 78 89 83
Vegetable sources (oil seed meals)

Peanut meals 49 88 - 91 83 88 78
Soybean meals 46 83 - 87 91 92 82
Cottonseed meal 43 61 - 76 67 73 73
Animal sources
Blood meal 88 82-92 86 91 76
Fish meal 66 86 - 90 88 92 73
Meat meal 60 75 - 80 79 85 58
Feather meal 87 36 - 77 66 76 59
FATS:
G.I Tract Region Enzyme (or
secretion)
Substrate End Product pH
Mouth Saliva Lubricates and softens food
Crop Mucus Lubricates and softens food 4.5
Stomach (Gizzard
and proventiculus)
HCI Lowers stomach pH 2.5
Lipase Fats Fatty acids, mono-
glycerides and glycerol
Duodenum and
Jejunum
Bile Fats Emulsification
Lipase Fats Fatty acids, mono-
glycerides and glycerol
5.8
Cholesterol
esterase
Fatty acid -
cholesterol
esters
Fatty acid, cholesterol to
6.6
Digestibility Metabolizable energy (kcal/kg)

Fat Type/Age 0-21d >21d 0-21d >21d
Tallow 80 86 7400 8000
Poultry Fat 88 97 8200 9000
Fish oil 92 97 8600 9000
Vegetable oil 95 99 8800 9200
Coconut oil 70 84 6500 7800
Palm oil 77 86 7200 8000
Vegetable
soapstock
84 87 7800 8100
Restaurant grease 87 96 8100 8900
Protease:
A protease (also termed peptidase or proteinase) is any enzyme that conducts proteolysis, that is, begins protein
catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming
the protein.
PROTEASES
Endopeptidases (proteinases) Exopeptidases(peptidases)
1. Serine proteases eg trypsin, chymotrypsin, subtilisin Aminopeptidases Carboxypeptidases
2.Cysteine proteaseseg papain, cathepsin B eg aminopeptidases I 1. Serine carboxypeptidases
3.Aspartic proteaseseg pepsin, chymosin Aminopeptidases II 2. Cysteine carboxypeptidases
4.Metalloproteases eg thermolysin, carboxypeptidase A 3. Aspartic carboxypeptidases
Classification of proteases (Source : Rao et al., 1998)
Proteases are currently classified into six broad groups:
1. Aspartate proteases
2. Cysteine proteases
3. Glutamic acid proteases
4. Metalloproteases
5. Serine proteases
6. Threonine proteases
The threonine and glutamic-acid proteases were not described until 1995 and 2004, respectively. The
mechanism used to cleave a peptide bond involves making an amino acid residue that has the cysteine and
threonine (proteases) or a water molecule (aspartic acid, metallo- and glutamic acid proteases) nucleophilic so
that it can attack the peptide carboxyl group. One way to make a nucleophile is by a catalytic triad, where a
histidine residue is used to activate serine, cysteine, or threonine as a nucleophile.
Within each of the broad groups proteases have been classified, by Rawlings and Barrett, into families of related
proteases. For example within the serine proteases families are labelled Sx where S denotes the serine catalytic
type and the x denotes the number of the family, for example S1 (chymotrypsins). An up to date classification of
proteases into families is found in the MEROPS database

Alternatively, proteases may be classified by the optimal pH in which they are active:
Acid proteases
Neutral proteases involved in type 1 hypersensitivity. Here, it is released by mast cells and causes
activation of complement and kinins.[3] This group includes the calpains.
Basic proteases (or alkaline proteases)
Proteases [serine protease (EC. 3.4.21), cysteine (thiol) protease (EC 3.4.22), aspartic proteases (EC 3.4.23) and
metallo-protease (EC 3.4.24)] constitute one of the most important groups of industrial enzymes, accounting for
about 60% of the total enzyme market.
Among the various proteases, bacterial proteases are the most significant, compared with animal and fungal
proteases and among bacteria, Bacillus sp are specific producers of extra-cellular proteases
Alkaline proteases
Strains of Bacillus, Steptomyces, Aspergillus are the major producers of alkaline proteases.
Neutral proteases
Neutral proteases are produced by bacteria (Clostridium histolyticum, Streptococcus spp., Bacillus subtilis, B.
cereus, B. megaterium, B. stearothermophilus, B. thuringiensis, B. pumilus, B. polymyxa, B. licheniformis, B.
amyloliquefaciens. B. stearothermophilus, Pseudomonas aeruginosa, Streptomyces griseus) and fungi
(Aspergillus oryzae, A. sojae, Penicillium spp., Pericularia oryzae). These microbial neutral proteases are either
cysteine or metalloproteases.
Acid Proteases
Alcaligens, Bacillus, Corynebacterium, Lactobacillus, Pseudomonas, Serratia, Streptococcus, and Streptomyces
are the bacteria, and Aspergillus, Candida, Coriolus, Endothia, Endomophthora, Irpex, Mucor,
Penicillium¸Rhizopus, Sclerotium, and Torulopsis are the some of the fungi producing rennin like proteases which
find applications in cheese processing.
Adjuvant for Protease:
Protease activity was stimulated by Mn
2+
and Ca
+2
. Several results suggest that these metal ions apparently
protected the enzyme against thermal denaturation and played a vital role in maintaining the active
conformation of the enzyme at higher temperatures.
(Beg, Q.K.; Gupta, R. Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline
protease from Bacillus mojavensis. Enz. and Microbial Techn., 32: 294-304, 2003.).
Similar effects of Mn
2+
on the activity of proteases were also observed by Rahman et al. , and by Manachini et al.
Protease Inhibitors
The activity of proteases is inhibited by protease inhibitors. One example of protease inhibitors is the serpin
super family, which includes alpha 1-antitrypsin, C1-inhibitor, antithrombin, alpha 1-antichymotrypsin,
plasminogen activator inhibitor-1, and neuroserpin.
Natural protease inhibitors include the family of lipocalin proteins, which play a role in cell regulation and
differentiation. Lipophilic ligands, attached to lipocalin proteins, have been found to possess tumor protease
inhibiting properties. The natural protease inhibitors are not to be confused with the protease inhibitors used in
antiretroviral therapy. Some viruses, with HIV/AIDS among them, depend on proteases in their reproductive
cycle. Thus, protease inhibitors are developed as antiviral means.
Degradation of Protease

Proteases, being themselves proteins, are known to be cleaved by other protease molecules, sometimes of the
same variety. This may be an important method of regulation of protease activity.
Protease research
The field of protease research is enormous. Barrett and Rawlings estimated that approximately 8001 papers
related to this field are published each year.
Protease Bonds with alpha 2-macroglobulin to support immune function when taken on an empty stomach.
Protease is responsible for digesting proteins in food, which is probably one of the most difficult substances to
metabolize. Because of this, protease is considered to be one of the most important enzymes that we have. If
the digestive process is incomplete, undigested protein can wind up in the circulatory system, as well as in other
parts of the body.
When protease is present in higher quantities, it can help to clean up the body by removing the unwanted
protein from the circulatory system. This will help to clean up the blood stream, and restore the energy and
balance.
One of the tricks of an invading organism is to wrap itself in a large protein shell that the body would view as
being "normal". Large amounts of protease can help remove this protein shell, and allow the body`s defense
mechanisms to go into action. With the protective barrier down, the immune system can step in and destroy the
invading organism.
Protease refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown) peptide bonds of
proteins. They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyze
various peptide bonds. Each type of protease has a specific kind of peptide bonds it breaks. Examples of
proteases include: fungal protease, pepsin, trypsin, chymotrypsin, papain, bromelain, and subtilisin.
Proteolytic enzymes are very important in digestion as they breakdown the protein foods to liberate the amino
acids needed by the body. Additionally, proteolytic enzymes have been used for a long time in various forms of
therapy. Their use in medicine is gaining more and more attention as several clinical studies are indicating their
benefits in oncology, inflammatory conditions and immune regulation.
Contrary to old beliefs, several studies have shown that orally ingested enzymes can bypass the conditions of
the GI tract and be absorbed into the blood stream while still maintaining their enzymatic activity.
Commercially, proteases are produced in highly controlled aseptic conditions for food supplementation and
systemic enzyme therapy. The organisms most often used are Aspergillus niger and oryzae.
Measured in HUT (Hemoglobin Units in a Tyrosine Base).
Gastrointestinal section pH Residence time/minutes
Crop 4,5-5,3 45
Proventriculus, gizzard 2,0-4,5 70
Ileum 5,6-7,9 160-200
Caecum 5,8-6,8 120
Colon, rectum 6,3-7,7 30-50
pH and residence time of feed in gastrointestinal tract
PROTEASES USED IN POULTRY
Following Data is available relating ProAct of DSM.

The protease (ProAct®, DSM) is an alkaline serine protease derived from Nocardiopsis prasina and the
production strain Bacillus licheniformis. Pepsin stability experiments find that 97% of this protease remains
intact and active after being exposed to pepsin for 1.5 hrs, pH 3 and at 40oC (Novozymes Internal Reports).
The protease was found to be effective across a wide range of peptide bonds and considered to be a non-
specific protease (Fischer et al., 2009. This protease is characterized by an optimal pH range that begins at pH of
5-6, and continues into the area of alkalinity. This range in operating pH complements existing endogenous
proteases such as pepsin and others that operate optimally in an acidic pH. Carvalho et al. (2009 a and b) and
Bertechini et al. (2009 a and b) presented ileal digestibility results with this protease.
This work confirms the ability of this protease to improve ingredient protein digestibility.
These researchers used the NFE diet or basal diet substitution methods to determine amino acid digestibility in
different ingredients. This method is patterned after the procedure developed by Matterson et al. (1965) in
which birds were fed a reference diet with significant portions being replaced by test ingredients. Across all
ingredients tested, corn, soybean meal, full fat soybean meal and meat and bone meal, the protease
improved amino acid digestibility. The improvement over the non-protease control
ranged from about 2% to 14%.
Apparent essential amino acid digestibility improvement in different diet with 200 ppm ProAct® protease.
Adapted from Favero et al., 2009, Maiorka et al., 2009 and Vila and Broz, 2008.
The digestibility improvement across essential amino acid was approximately 4% with the use
of 200 ppm ProAct® protease.
In a study done at the University of Maryland in 2009 (Angel, 2010) with Ross 708 straight run broilers were fed
corn soy diets with graded concentrations of this protease from 7 to 22 d of age in batteries. Six diets were fed.
A positive control (PC) that was formulated based on AgriStats (2008) met or exceeded all NRC (1994) nutrient
recommendations. The PC diet contained 22.5% crude protein. All other diets were negative control diets with
the protease (0, 100, 200, 400, 800 ppm). The negative control diet contained 20.5% crude protein and a 9%
reduction in the concentration of Lys, Met, TSAA and Thr was forced into the diet. All diets were isocaloric. The
content of the distal ileum was removed with water, freeze dried, and ground and analyzed for marker, nitrogen
and amino acids. Analysis of the diets confirmed levels close to formulated levels. There was no clear dose
response for any of the essential amino acids. Overall, the improvement in essential amino acid digestibility due
to the protease was 4.8% when 200 ppm was used with the greatest impact seen in threonine. The non essential
amino acids were improved by an average of 3.72% with Asp and Ser being the most positively affected Across
several studies, threonine digestibility is improved to the greatest extent with the ProAct® protease. This is true
for most individual ingredients, as well as corn/SBM diets fed for ileal amino acid digestibility determinations.

DATA ON PROTIGEST
1. Source of origin (wheather Fungal or Bacterial)
Fungal
2. Type of Fermentation used for harvesting
Solid state fermentation
3. Nature of the product (Acid or Alkaline)
Mix of Acid, Neutral and Alkali
4. Which nature Protease is suitable for mixing in poultry feeds.
Proteases perform a variety of roles in biology. These enzymes function in important physiological
processes, including homeostasis, apoptosis, signal transduction, reproduction and immunity (Hedstrom,
2002). In addition, proteases are involved in blood coagulation and wound healing. To accomplish a wide
range of tasks, over 500 proteases exist in humans; a similar number is present in other organisms.
From a nutritionist’s perspective, the hydrolysis of proteins to individual amino acids and peptides in the
intestinal tract is a key function for proteases. Several intestinal proteases exist, and comprise a “protease
system” in the intestinal tract for the utilization of various protein sources.
Hence a suitable mix of several Proteases is required.
5. If it is a combination, what is the RATIO of acid type to alkaline type.
57.5% Acidic Protease(pH 4.0), 37.5% Neutral Protease (pH 6.0) and 5% Alkaline (pH 8.0) Protease
combination is found to be the best option .
6. Degree/ Extent of IN-VITRO hydrolysis of feed proteins and the
probable time- period taken.
Depends on the source of protein, will hydrolyze up to 80% of soya protein
Passage of hydrochloric acid, produced in the proventriculus, and peptides through the proventriculus and
gizzard into the duodenum stimulates the release of the hormones secretin and pancreozymin from the mucosa
of the duodenum (Rothman and Wells, 1967). These hormones promote the secretion of pancreatic juice
containing a number of enzymes and bicarbonate ions. The production of an alkaline solution quickly neutralizes
the acid entering the duodenum (Barash et al., 1993). Small intestinal enzymes function best at pHs close to
neutral or slight below neutral and thus, insufficient alkaline bile, lowers enzyme activity in the intestine
(Zentler-Munro, 1985).
Pancreatic proteases are secreted from the pancreas only in the form of zymogens. All known cellular proteases
are synthesized as zymogens, or the inactive precursor, toprevent unwanted protein degradation at the point of
origin or thereabouts (Kahn andJames, 1998). The conversion of the zymogens to the active protease requires
low pH(autocatalysis) or limited proteolysis. Primary pancreatic zymogens are trypsinogen, chymotrypsinogens
A and B, proelastase, and procarboxypeptidases A and B. Trypsin is activated after being attacked by
enterokinase, found in the brush border membrane. The active trypsin then hydrolyses bonds in the other
zymogens, releasing the active enzymes.

From a practical nutritionist’s standpoint, the value of ingredients as a protein (amino acid) source generally
comes down to the total amino acids it contains, the ratio of amino acids and availability of the amino acids
(aside from cost, ingredient accessibility, etc.).
Numerous studies and tables on protein and amino acid availability (the digestibility, absorption and utilization
by the animal) have been published. The common thread that runs through such summaries is that ingredient
improvements can yet be made in digestibility of protein.
In one large scale study (Ravindran et al., 2005), apparent ileal digestibility was determined for various
ingredients for broilers. For cereals, the overall amino acid digestibility coefficient for eight samples of corn was
0.81 (range 0.77 to 0.85). For soybean meal, the digestibility coefficient was 0.82 (range 0.81 to 0.83). In
particular, the groups of meat and bone meal (avg 0.62) and meat meal samples (avg digestibility coefficient
0.65) showed low amino acid digestibility values. This was accompanied by marked variation.
Similarly, University of Illinois work has reported a number of studies on amino acid digestibility of ingredients.
Low values and high variation for meat and bone meals is typical
(Parson et al., 1997; Parsons et al., 1998).
More recent collaborative work with The Ohio State University, Purdue University and University of Illinois
further verifies such data
(Adedokun et al., 2007).
(http://www.dsm.com/en_US/downloads/dnpna/Proteases_Potential_for_Use_in_Poultry_Nutrition.pdf)
7. Stability during storage and against PELLETIZATION.
Storage stability 1 year, tolerant to pellet temperature of 80oC for 1 min. and cooling time of 5 min
8. Maximum CONCENTRATION/ACTIVITY per GRAM
2,00,000 HUT/g
9. ANY OTHER SPECIFICATIONS?
Light brown colored powder
10. HOW IT DIFFERS WITH OTHER ENZYMES IN THE MARKET?
Thermo-stable and broad spectrum PROTIGEST aggressively works in the entire GI tract irrespective of pH
variations.
PROTIGEST complements poultry’s endogenous enzymes.
Handling and Safety
Enzymes are proteins, and as is the case with all proteins, people can have allergic reactions when exposed to
them. Most enzymes are in a dry form, so airborne contamination and exposure is possible. It is important that
workers be aware of proper handling techniques.
However, if exposure is a concern based upon the specific manufacturing environment, preparations are
available that are either in a liquid form, or specially processed to minimize the dust
SUGGESTED LEVEL AND METHOD OF USAGE:
50-100G PER MT FEED REGULARLY
Ref:

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