DFM to replace Enzymes for use in Animal feeds

1. ENZYMIX
ENZYMES WITH DIRECT FED ENZYME SECRETING MICROBES
INTRODUCTION
Improved animal health and performance always has been the goal of people associated
with livestock production. Consequently, any feedstuff, feed additive, drug or other
compound that is capable of enhancing animal health or performance will interest
producers, veterinarians, and animal nutritionists. Several compounds have been used
to improve animal performance either by manipulation of the rumen environment (e.g.,
sodium bicarbonate) or by directly altering the composition and metabolic activities of
rumen microorganisms (e.g., ionophores).
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.
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-glucans and arabinoxylans) are non digestible and
form high-molecular-weight viscous aggregates in the gastrointestinal tract.
They
• Affect the digestive enzymes.
• Cause endogenous losses

2. • Reduce the rate of passage.
• Stimulates pathogenic microbial proliferation.
Enzymes such as xylanase or ß-glucanase into diets having ANF can effectively
decrease viscosity and consequently reduce the anti nutritional effect of NSP.
Cellulase, ß-Glucanase, Xylanase, and Pectinase can degrade plant origin cell wall
polymers.
Amylase can increase the gut absorption levels of starches.
Hemi Cellulase can degrade the difficult fiber.
Protease can degrade the proteins.
Lipase can degrade the lipids.
Phytase helps in solubilising the phyto phosphorous.
Tannases can degrade the plant toxins.
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

3. 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

4. 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:

5. 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
Cellulase
Breaks down cellulose and chitin

6. (chitin is cellulose like fiber found in the cell wall of candida).
Cellulases acts on cellulose molecules by hydrolysing the beta-1, 4 glycosidic linkages.
They largely produces cellobiose, which can ultimately yield glucose units, depending on
the characteristic of the enzyme.
It helps free nutrients in both fruits and vegetables.
Measured in CU (Cellulase Units).
Activity of Enzyme:
One activity u/mg (u/ml) of cellulase is defined as that quantity of enzyme that can
liberate 1g glucose in one minute at pH of 4.8.
Standard Product: 8000 u/g
Use the preparation between pH of 4.0~5.0,
Generally speaking, adding the preparation 50g to 1t dry materials will play well.
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

7. 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).
Lipase
Lipase is an enzyme necessary for the absorption and digestion of nutrients in the
intestines. This digestive enzyme is responsible for breaking down lipids (fats), in
particular triglycerides, which are fatty substances in the body that come from fat in the
diet. Once broken down into smaller components, triglycerides are more easily absorbed
in the intestines. Lipase is primarily produced in the pancreas but is also produced in the
mouth and stomach. Most people produce sufficient amounts of pancreatic lipase.
Along with lipase, the pancreas secretes insulin and glucagon, hormones that the body
needs to break down sugar in the bloodstream. Other pancreatic enzymes include
amylase, which breaks down amylose (a form of starch) into its sugar building blocks,
and protease, which breaks down protein into single amino acids.
Source:
Lipase, monoacylglycerol:
Penicillium camembertii
Lipase, triacylglycerol:
Aspergillus niger
Aspergillus oryzae
Aspergillus oryzae, containing the gene for Lipase, triacylglycerol isolated from
Fusarium oxysporum
Aspergillus oryzae, containing the gene for Lipase, triacylglycerol isolated from
Humicola lanuginosa
Aspergillus oryzae, containing the gene for Lipase, triacylglycerol isolated from
Rhizomucor miehei
Rhizopus arrhizus
Rhizomucor miehei
Rhizophus niveus
Rhizophus oryzae
Uses
In general, lipase supplements are thought to help the body absorb food more easily,
keeping nutrients at appropriate, healthy levels throughout the body. Studies suggest
that they may also be helpful for the following conditions:
Celiac Disease
Pancreatic enzymes have been most studied as part of the treatment for celiac disease.
Celiac disease is a condition in which dietary gluten causes damage to the intestinal
tract. Symptoms include abdominal pain, weight loss, and fatigue. People with celiac
disease must consume a life-long gluten-free diet. Lipase, along with other pancreatic
enzymes, may help in the treatment of this condition by enhancing the benefit of a
gluten-free diet. In a study of 40 children with celiac disease, for example, those who
received pancreatic enzyme therapy (including lipase) demonstrated a modest increase
in weight compared to those who received placebo. The improvement in weight occurred
within the first month of use; taking the pancreatic enzyme supplements for an additional
month did not lead to more weight gain.

8. Indigestion
In a small study including 18 subjects, supplements containing lipase and other
pancreatic enzymes were found to reduce bloating, gas, and fullness following a high-fat
meal. Given that these symptoms are commonly associated with irritable bowel
syndrome, some with this condition may experience improvement with use of pancreatic
enzymes.
Other
Although scientific evidence is lacking, lipase has been used by trained clinicians to treat
food allergies, cystic fibroris, and autoimmune disorders, such as rheumatoid arthritis
and lupus.
Dietary Sources
Lipase is produced primarily in the pancreas and is not found in food.
Available Forms
Lipase supplements are usually derived from animal enzymes, although plant sources of
lipase and other digestive enzymes have become increasingly popular. Lipase may be
taken in combination with protease and amylase enzymes.
Pectinase
Breaks down carbohydrates, such as pectin found in many fruits and vegetables.
Measured in AJDU
Activity of Enzyme: One activity u/mg(u/ml) of pectinase is defined as that quantity of
enzyme that can liberate 1 galacturonic acid in one minute at pH of 3.5,
Standard pectinase :1200000 u/ml min
Optimum pH of 3.2~5.0,
The activity of enzymes retains more than 95% when stored at the temperature of 25
Deg C; upto 3 months.
Xylanase
Xylanase (EC 3.2.1.8) is the name given to a class of enzymes which degrade the linear
polysaccharide beta-1,4-xylan into xylose, thus breaking down hemicellulose, which is a
major component of the cell wall of plants.
As such, it plays a major role in the digestive system of herbivorous micro-organisms
(mammals, conversely, do not produce xylanase).
Additionally, xylanases are present in fungi for the degradation of plant matter into
usable nutrients.
Commercial applications for xylanase include the chlorine-free bleaching of wood pulp in
the papermaking process, and the increased digestibility of silage (in this aspect, it is
also used for fermentative composting).
In the future, xylanase may be used for the production of biofuel from unusable plant
material.
DIRECT FED MICROBES

9. Many studies have reported the effective usefulness of DFM in
• Detoxifying
• Enhancing the immunity System,
• Improving F C R
• Reducing the Diarrhoea,
The epithelial cells of the intestine are covered by a protective layer of mucus, which is a
complex mixture of glycoproteins and glycolipids with the large glycoprotein mucin being
the main component. Ability of probiotic bacteria to adhere to the intestinal mucus is
considered important for transient colonization, antagonism against pathogens,
modulation of the immune system, and enhanced healing of damaged gastric mucosa
Actinomyces, Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Prevotella ruminicola,
Ruminococcus albus and Ruminococcus flavefaciens secrete fiber degrading enzymes.
Piromyces rhizinflata secrets carboxymethyl cellulase enzymes.
Bacillus subtilis, Enterococcus diacetylactis, Lactobacillus acidophilus and other
Lactobacillus species like L. casei
• Would decrease or prevent intestinal establishment of pathogenic
microorganisms (Vandevoorde et al., 1991)
• Would re-colonize a “stressed” intestinal environment and return gut function to
normal more quickly
• have reduced incidence of diarrhea (Beecham et al., 1977)
• reduced counts of intestinal coliform bacteria (Bruce et al., 1979).
Propionibacteria may be beneficial if inoculated into the rumen (Kung et al., 1991)
because higher concentrations of ruminal propionate would be absorbed into the blood
and converted to glucose by the liver of the host animal
Acetobacter Xylinum
• Degrades levulinic acid.
• Production of Dienes, 7 – Cyano Steroids.
• Useful in steroid conversion.
• Useful in dehydroxylation of cholic acids.
• Produces 5 nucleotides by cell culture on hydrocarbons.
• Produces 3 keto 1,4 Steroids.
Aspergillus niger
• Produces Sachharifying enzymes, glucoamylase, large amounts of maltase,
and less amounts of amylase.
• Produces Beta galactesidase.
Aspergillus Oryzae
• Produces Amylase, Protease, Tannase.
• Does not produce aflatoxins.
• when fed to lactating ruminants, it is found to result in an average increase in
milk production of about 0.45 kg (1.01 lb.) of milk per day.

10. B lechiniformis
• Provides Bacitracin.
B. polymixa
• Possesses unusual characteristic to fix nitrogen under anaerobic conditions.
• Solubilises Phosphorous.
• Produces polymixin a polypeptide antibiotic, which possess the ability to
damage cell membrane structure.
• Asymbiotic Nitrogen fixer.
• Gram negative.
• Produces 2,3 Butanediol used as solvent, humectant, chemical intermediate
B. megaterium
• Solubilises Phosphorous in higher pH of the medium.
B subtilis
The nutritive qualities of microbial communities that abound the water medium as also
possible enhancement of nutritive values of artificial feeds through microbial processing
when assessed gave the following results. The crude protein levels (%ges. on dry
matter Basis) Bacillus subtilis, Aspergillus flavus, Plankton, Wheat Bran – GNC Mixture,
Fermented Bran-Cake , processed Water Hyacinth were 43.63, 47.27. 42.91, 29.09,
36.37 and 14.5respectively. Corresponding Energy values (Kcal / gm dry weight) are
2.98, 4.03, 3.78,3.02, 3.68 and 3.01 respectively. Specific growth rates of the carp fry
fed on the above ranged between 3.40 to 0.02. This indicates the significance of
microbial communities as triophic components and showed possibilities of their better
utilization for carp rearing through medium enrichment and diet incorporation.
• Gram positive.Thermoduric.
• Solubilises Phosphorous.
• Degrades proteins.(Proteolytic) and Carbon (amylolytic).
• Produces amylases and protease enzymes. Used to modify starches, in sizing
paper and textiles.
• Can remain active in excreta resulting in less odor, faster decomposition, and in
reduction of solids.
• Produces bacitracin, which interferes with regeneration of the monophosphate
form of bactoprenol from the pyrophosphate form.
Lactic Acid Bacillus
( Formerly known as Lacto Bacillus Sporogenes )
• Proved effective in lowering LDL Cholesterol.
• Provides an excellent preventative effect against various diseases of the
intestine.
• Increases production of Rotefiers.
• Limits the proliferation of pathogens in rotifiers.
• Provides a source of immunostimulant .
• Useful in the cases of non specific vaginitis / leucorrhoea
• Safe during lactation and in elderly.
Lactobacillus delbrueckii

11. • Immune Stimulation
• Produces Lactic Acid
L lactis
• Reduces the ability of pathogenic bacteria to grow and cause infection.
• Especially effective against Listeria monorytogenes, which causes severe food
poisoning.
L acidophillus
• Produces extremely effective natural antibiotic substances that can inhibit
11 known disease causing bacteria.
• It has also been proven to inhibit yeast infections.
• Helps in cases of chronic constipation and diarrhea by replacing
undesirable intestinal organisms.
• Helps in the cases of food poisoning.
• Lowers levels of LDL Cholesterol.
• Found to alleviate intestinal disorders, principle being that the ingestion of
large numbers of the lactobacilli may result in replacement of undesirable
intestinal organisms by harmless and beneficial organisms, a concept
first proposed by the Russian Bacteriologist Metchinikoff in the early days
of bacteriology. The implantation of the lactobacilli seems to depend on
ingestion of large number of organisms and on supplying a suitable
carbohydrate such as lactose that is not readily absorbed by the body but
can be easily used by the organism.
• Aids in nutrient uptake.
• Fights Candida overgrowth.
• Controls effectively E Coli and Staphylococcus aureus.
Lactobacillus reutri
• Possess resistance to bile salt and acid.
• Possess the capacity to break down soluble carboxymethyl cellulose, ß-
glucan, or xylan. Possess high adhesion efficiency to mucin and mucus.
• Produces an autoaggregation-promoting protein.
• Produces antimicrobial substance reuterin.
• Produces fibrolytic enzymes.
Effective in enhancing the growth and development of poultry subject to stressors similar
to those likely to be encountered under commercial production conditions. L.reuri is the
only species of known microorganism which can produce reuterin, a newly discovered
antimicrobial agent which inhibits growth of other intestinal pathogenic microbes such as
Salmonella, Listeria, Escherichia.
In addition, there is evidence that L. reuteri formulations effectively counteract weight
losses caused by disease associated stress and competitively reduce Salmonella. In
another trial, the result showed that L. reuteri and whey in the diet reduced mortality
from natural causes and from mortally induced by a Salmonela challenge.
Also poultry treated with L. reuteri and whey were heavier than non treated animals.

12. Another conclusion was that L. reuteri and whey in the diets of turkey poults appeared
beneficial in the control of caecal Salmonella populations.
Lyophilized L. reuteri exhibit excellent long term viability.
It has been shown that early colonization with L. reuteri contributes to the development
of healthy birds, possibly because of the effect it has on the development of the gut
mucosal tissues.
Transitory cold stress during the first 48 hours after the placement of the birds causes a
growth depression through 20 days of age. Addition of L. reuteri to the diet of these cold
stressed birds plays a significant role in overcoming the suppressive effects.
Reduced variability in body weights of the birds was observed with the feeding of L.
reuteri resulting in more uniform flock. Controls Cryptosporidium parvum infection.
Beneficial in treatment of watery diarrhea.
Sachromyces cerevisiae 3090
• Has ability to improve milk production consistently.
• Helps in oligosaccharide transfer in microsomes.
• May also stimulate rumen fermentation by scavenging excess oxygen from
the rumen (Newbold et al., 1996).
• May have a buffering effect in the rumen by mediating the sharp drops in
rumen ph, which follows feeding.
• May help to buffer excess lactic acid production when ruminants are fed high
concentrate diets.
• Possess invertase activity.
• Produces arginase.
Most investigators agree that yeast culture supplementation strategies can have
measurable effects on ruminal fermentations, and a number of beneficial changes in
digestion have been noted. Studies in several laboratories have demonstrated that yeast
culture supplementation can influence digestive processes in the rumen (Williams and
Newbold, 1990; Dawson, 1992; Newbold et al., 1996; Wallace 1996). Typically, the total
extent of dry matter digestion was not drastically altered. However, the initial rate of
digestion is readily influenced by the addition of live yeast preparations to the diets of
ruminants . This is a characteristic of yeast supplementation that has been measured in
both in vitro (Dawson and Hopkins, 1991) and in vivo studies (Williams and Newbold,
1990; Smith et al., 1993; Kumar et al., 1997). Since feed intake is often considered to be
a function of the initial rates of fiber digestion, early stimulation of ruminal activity can be
expected to have a major impact on feed consumption and can provide a driving force
for improved animal performance. Such studies suggest an important role for yeast
culture supplementation in digestion of animals maintained on high forage diets.
Other studies have demonstrated a role for yeast culture in stabilizing ruminal
fermentations and in addressing ruminal disorders. Williams et al. (1991) demonstrated
the beneficial effects of the viable yeast culture, on lactic acid concentrations in the
rumen in high concentrate diets. In animals fed high energy diets, decreased lactic acid
concentrations are associated with higher ruminal pH and are characteristic of much
more stable ruminal fermentation. These alterations in ruminal fermentation can be
expected to provide for improved digestion and could also be reflected in improved
intake and production. The ability of yeast to prevent the accumulation of lactic acid in

13. the rumen suggests a role for viable yeast in overcoming ruminal dysfunctions
associated with the use of high energy diets used in both high-producing dairy and fast-
growing beef cattle.
Several lines of evidence suggest that yeast culture supplementation can beneficially
alter nitrogen metabolism in the rumen . This is reflected in lower ruminal ammonia
concentrations observed in animals receiving yeast supplements and is consistent with
observed increases in the concentrations of bacteria in the rumen. In addition, these
changes are reflected in an increased flow of bacterial nitrogen to the small intestines
(Erasmus et al., 1992). Altered nitrogen flow has also been associated with shifts in the
basic amino acid flow out of the rumen. The beneficial increase in the flow of microbial
protein from the rumen is consistent with models that suggest stimulation of microbial
growth in the rumen and more efficient conversion of ammonia nitrogen into microbial
protein. Since microbial protein is often used to drive protein synthesis in high-producing
ruminants, these observations suggest a role for specific yeast culture supplements in
stimulating protein synthesis in both beef and dairy production systems.
Many investigators have attributed the beneficial effects of yeast culture directly to
changes in the ruminal fermentation and to changes in the microbial population in the
digestive tract . The ability of specific yeast culture preparations to stimulate the growth
of ruminal bacteria and to increase the concentrations of specific groups of beneficial
bacteria in the rumen has been well documented. Increased concentrations of the total
anaerobic bacteria and of cellulolytic bacteria have been one of the most consistently
measured responses to yeast culture in the rumen .However, other studies have also
suggested that yeast culture preparations can enhance the growth of lactic acid-utilizing
bacteria, proteolytic bacteria and bacteria that convert molecular hydrogen to acetate in
the rumen. In addition, yeast preparations have been shown to enhance the activities of
fiber-digesting fungi in the rumen. Increased concentrations of beneficial microorganisms
and enhanced microbial activities can be expected to lead to enhanced digestive
processes and the destruction of metabolic intermediates that can result in ruminal
dysfunction. The ability of yeast to stimulate specific groups of bacteria is consistent with
many of the other physiological and metabolic effects of yeast observed in the rumen
and can explain enhanced protein synthesis, improved ruminal stability and improved
microbial protein synthesis.
Despite the basic understanding of some of the beneficial effects of yeast cultures on the
bacterial population in the rumen, the physiological basis for the enhanced microbial
growth has not been completely described. A number of specific hypothetical
biochemical mechanisms have been developed to explain the stimulatory effects of
yeast cultures in the rumen (Dawson and Girard, 1997). Some of these have been
based on the ability of yeast to provide important nutrients or nutritional cofactors that
stimulate microbial activities while others suggest that the ability of yeast to control the
oxygen level in the ruminal environment is important. These kinds of models have many
attractive features but are individually limited in their ability to explain all of the effects
associated with yeast supplementation in the rumen.
However, recent studies have suggested that more basic mechanisms are involved in
the overall stimulation of beneficial ruminal bacteria. These studies have resulted in the
isolation of a group of small compounds that stimulate bacteria to enter into logarithmic
growth and thus stimulate microbial activities. Some of the basic characteristics of these
stimulatory compounds are consistent with the basic characteristics of small biologically

14. active peptides. The stimulatory activities of these small peptides has been
demonstrated in studies with pure cultures of ruminal bacteria. Synthetic tryptophan
containing peptides have also been shown to bring about similar stimulatory effects and
have also been shown to stimulate the growth of representative fiber-digesting bacteria
from the rumen. These stimulatory activities were not associated with individual amino
acids . Stimulatory activities occurred at concentrations well below those that would
suggest that these compounds are limiting nutrients. Instead, they appear to serve as
metabolic triggers that stimulate beneficial ruminal bacteria to enter into an exponential
growth phase. This stimulatory activity toward specific strains of ruminal bacteria can
explain many of the observed effects of yeast culture in the rumen.
It is important to recognize that stimulatory effects of viable yeast in the rumen only
address some of the beneficial effects of yeast in animal production systems. Research
in this area has resulted in a number of new concepts that have evolved into useful
animal supplements. Currently yeast cell wall products are available that prevent
colonization by pathogenic microorganisms in the gut, alter the composition of microbial
populations in the intestinal tract, modulate immune function and alter the structure of
the gut wall. These developments are all the result of in depth study of yeast culture
supplementation and are currently used to enhance the performance of both ruminant
and non-ruminant animals.
Trichoderma reesei
• Produces glucose by enzymatic hydrolysation of cellulose.
• Produces Cellulase.
• Produces D glucanase.
• Produces cell wall lytic enzymes.
When compared to Enzymes, DFM are cost effective and not cumbersome.
However one hurdle in using DFM is that prior to reaching the intestinal tract, these DFM
must first survive transit through the stomach, where secretion of gastric acid represents
a primary defense mechanism against the majority of ingested microorganisms. BIOOPS
has overcome this problem successfully by adopting specific strains that can overcome
this problem.
Another problem of using DFM is that now a days almost all feeds are pelletised at about
105 Deg C, which temperature may damage the DFM. Hence tolerance of DFM to heat
is becoming an important factor.
In general, most yeast, Lactobacillus, Bifidobacterium, and Streptococcus are destroyed
by heat during pelleting. In contrast, bacilli form stable endospores when conditions for
growth are unfavorable and are very resistant to heat, pH, moisture and disinfectants.
BIOOPS has solved this problem by adopting procedures to improve the heat tolerance
of the specific strains used.
Organism End Products or Potential Use
Bacillus subtilis amylase, protease
Bifidobacterium bifidum ureases, lactic acid, formic acid
L. lactis amylase, hydrogen peroxide, proteases
Lactobacillus acidophilus lactic acid, acidophilin, glycosidases

15. Pediococsus acidilactici pediocin (bacteriocin)
Propionibacteria sp. Ruminal lactate utilizer, propionate producer
Propionibacterium thoenii propionicin PLG-1 (bacteriocin)
Bacillus polymyxa Polymixin B Antifungal Peptide
COMPOSITION OF ENZYMIX
DFM < 1000 Million CFU/g
SALIENT FEATURES OF ENZYMIX
• Higher quality end product from cleaner eggs or reduced carcass downgrades
• Improved environment
• improves the digestibility of the feed
• Lower feed costs
• More uniform pigs and birds
• Proven value in antibiotic growth promoter free nutrition
• Provides feed manufacturing with the opportunity to choose low cost feeding
stuff to replace high cost feeding stuff.
• Provides feed manufacturing with the opportunity to reduce feed costs
SUGGESTED LEVEL OF INCLUSION:
Generally speaking, adding ENZYMIX 75- 150 g to 1M T dry materials will play well. But
you should reconfirmed suitable dosage depending on your bench-scale experiment
results.
OTHER MATTERS OF IMPORTANCE
Use the preparation between pH of 3.8~5.0,
The activity of enzymes retains more than 95% when stored at the temperature of 25
Deg C upto 3 months
WITHDRAWAL PERIOD
Not necessary
STORAGE:
All Enzymes and DFM should be kept away from moisture, excess heat, and light
Ref:
1. Ahmed, F. E. 2003. Genetically modified probiotics in foods. Trends Biotechnol. 21:491-497.
2. Applied and Environmental Microbiology, November 2005, p. 6769-6775, Vol. 71, No. 110099-2240/05/$08.00+0
doi:10.1128/AEM.71.11.6769-6775.2005
3. Baran, M., and V. Kmet. 1987. Effect of pectinase on rumen fermentation in sheep and lambs. Arch. Anim. Nutr. Berlin.
7/8:643.
4. Beauchemin, K. A., W. Z. Yang, and L. M. Rode. 1999. Effects of grain source and enzyme additive on site and extent of
nutrient digestion in dairy cows. J. Dairy Sci. 82:378-390.
5. Beauchemin, K., A., and L. M. Rode. 1996. Use of feed enzymes in ruminant nutrition. Proc. of the Canadian Society of
Animal Science Annual Meeting, Lethbridge, Alberta. Pp 103-140.
6. Beauchemin, K., A., L. M. Rode, and V.J.H. Sewalt. 1995. Fibrolytic enzymes increase fiber digestibility and growth rate
of steers fed dry forages. Can. J. Anim. Sci. 75:641-644.

16. 7. Bedford, M. R., and H. Schulze. 1998. Exogenous enzymes for pigs and poultry. Nutr. Res. Rev. 11:91-114.[CrossRef]
8. Beecham, T. J., J. V. Chambers, and M. D. Cunningham. 1977. Influence of Lactobacillus acidophilus on performance of
young dairy calves. J. Dairy Sci. 60(Suppl. 1):74. (Abstract)
9. Beharka, A. A., T. G. Nagaraja, and J. L. Morrill. 1991. Performance and ruminal development of young calves fed diets
with Aspergillus oryzae fermentation extracts. J. Dairy Sci. 74:4326-4336.
10. Berkow R, ed. The Merck Manual of Medical Information . Home Ed. Whitehouse Station, NJ: Merck Research
Laboratories; 1997.
11. Bruce, B. B., S. E. Gilliland, L. J. Bush, and T. E. Staley. 1979. Influence of feeding cells of Lactobacillus acidophilus on
the fecal flora of young dairy calves. Oklahoma Anim. Sci. Res. Rep. 207.
12. Carroccio A, Iacono G, Montalto G, et al. Pancreatic enzyme therapy in childhood celiac disease. A double-blind
prospective randomized study. Dig Dis Sci . 1995;40(12):2555-2560.
13. Cheng, K. J., S. S. Lee, H. D. Bae, and J. K. Ha. 1999. Industrial applications of rumen microbes. Asian-Australas. J.
Anim. Sci. 12:84-92.
14. Choct, A., and G. Annison. 1992. Anti-nutritive effect of wheat pentosans in broiler-chickens: role of viscosity and gut
microflora. Br. J. Poult. Sci. 33:821-834.
15. Dawson, K. A., and D. M. Hopkins. 1991. Differential effects of live yeast on the cellulolytic activities of anaerobic ruminal
bacteria. J. Anim. Sci. 69(Suppl. 1):531. (Abstract)
16. Dawson, K. A., K. E. Neuman, and J. A. Boling. 1990. Effects of microbial supplements containing yeast and lactobacilli
on roughage-fed ruminal microbial activities. J. Anim. Sci. 68:3392-3398.
17. Ehrmann, M. A., P. Kurzak, J. Bauer, and R. F. Vogel. 2002. Characterization of lactobacilli towards their use as probiotic
adjuncts in poultry. J. Appl. Microbiol. 92:966-975.[CrossRef][Medline]
18. Englyst, H. N., S. A. Bingham, S. A. Runswick, E. Collinson, and J. H. Cummings. 1989. Dietary fibre (non-starch
polysaccharides) in cereal products. J. Hum. Nutr. Diet. 2:253-271.
19. Feng, P., C. W. Hunt, W. E. Julien, K. Dickinson, and T. Moen. 1992. Effect of enzyme addition on in situ and in vitro
degradation of mature cool-season grass forage. J. Anim. Sci. 70(Suppl. 1):309. (Abstract)
20. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bact. 66: 365-378
21. Gusils, C., O. Oppezzo, R. Pizarro, and S. Gonzalez. 2003. Adhesion of probiotic lactobacilli to chick intestinal mucus.
Can. J. Microbiol. 49: 472-478.[CrossRef][Medline]
22. Heck AM; Yanovski JA; Calis KA. Orlistat, a new lipase inhibitor for the management of obesity. Pharmacother . 2000
Mar;20(3):270-279.
23. Hirstov, A., T. A. McAllister, and K. J. Cheng. 1998. Effect of dietary or abomasal supplementation of exogenous
polysaccharide-degrading enzymes on rumen fermentation and nutrient digestibility. J. Anim. Sci. 76:3146-3156.
24. Hutchenson, D. P., N. A. Cole, W. Keaton, G. Graham, R. Dunlap, and K. Pitman. 1980. The use of living, nonfreeze-
dried Lactobacillus acidophilus culture for receiving feedlot calves. Proc. West. Sec. Amer. Soc. Anim. Sci. 31:213.
(Abstract)
25. Jaquette, R. D., R. J. Dennis, J. A. Coalson, D. R. Ware, E. T. Manfredi, and P. L. Read. 1988. Effect of feeding viable
Lactobacillus acidophilus (BT1386) on the performance of lactating dairy cows. J. Dairy Sci. 71(Suppl. 1):219. (Abstract)
26. Jenny, B. F., H. J. Vandijk, and J. A. Collins. 1991 Performance and fecal flora of calves fed a Bacillus subtilis
concentrate. J. Dairy Sci. 74:1968-1973.
27. Jin, L. Z., Y. W. Ho, N. Abdullah, and S. Jalaludin. 1998. Acid and bile tolerance of Lactobacillus isolated from chicken
intestine. Lett. Appl. Microbiol. 27:183-185.[CrossRef][Medline]
28. Kerovuo, J., and S. Tynkkynen. 2000. Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755. Lett. Appl.
Microbiol. 30:325-329.
29. Konig, J., R. Grasser, H. Pikor, and K. Vogel. 2002. Determination of xylanase, ß-glucanase, and cellulase activity. Anal.
Bioanal. Chem. 374: 80-87.[CrossRef][Medline]
30. Kopency, J., M. Marounek, and K. Holub. 1987. Testing the suitability of the addition of Trichoderma viride cellulases to
feed rations for ruminants. Zivocisna Vyroba. 32:587.
31. Kung, L., Jr., and A. O. Hession. 1995. Altering rumen fermentation by microbial inoculation with lactate-utilizing
microorganisms. J. Anim. Sci. 73:250-256.
32. Kung, L., Jr., E. M. Kreck, R. S. Tung, A. O. Hession, A. C. Sheperd, M. A. Cohen, H. E. Swain, and J.A.Z. Leedle. 1996.
Effects of a live yeast culture and enzymes on in vitro ruminal fermentation and milk production of dairy cows. J. Dairy
Sci. 80:2045-2051.
33. Martin, S. A., and D. J. Nisbet. 1992. Effect of direct-fed microbials on rumen microbial fermentation. J. Dairy Sci.
75:1736-1744.
34. Mathlouthi, N., M. A. Mohamed, and M. Larbier. 2003. Effect of enzyme preparation containing xylanase and ß-
glucanase on performance of laying hens fed wheat/barley- or maize/soybean meal-based diets. Br. Poult. Sci. 44:60-66.
[CrossRef][Medline]
35. Newbold, C. J. 1995a. Microbial feed additives for ruminants. In: Biotechnology in Animal Feeds and Animal Feeding.
R. J. Wallace and A. Chesson (Eds.). VCH. New York. Pp. 259-278.
36. Newbold, C. J., R. J. Wallace, and F. M. McIntosh. 1996. Mode of action of the yeast Saccharomyces cerevisiae as a
feed additive for ruminants. Brit. J. Nutr. 76:249.

17. 37. Newbold, C. J., R. J. Wallace, X. B. Chen, and F. McIntosh. 1995b. Different strains of Saccharomyces cerevisiae differ
in their effects on ruminal bacterial numbers in vitro and in sheep. J. Anim. Sci. 73:1811-1818.
38. Orr, C. L., D. R. Ware, E. T. Manfredi, and D. P. Hutchenson. 1988. The effect of continuous feeding of Lactobacillus
acidophilus strain BT1386 on gain and feed efficiency of feeder calves. J. Anim. Sci. 66(Suppl. 1): 460. (Abstract)
39. Patterson, J. A., and K. M. Burkholder. 2003. Application of prebiotics and probiotics in poultry production. Poult. Sci.
82:627-631.[Abstract/Free Full Text]
40. Physicians' Desk Reference . 55th ed. Montvale, NJ: Medical Economics Company, Inc.; 2001.
41. Reid, G., and R. Friendship. 2002. Alternatives to antibiotic use; probiotics for the gut. Anim. Biotechnol. 13:97-112.
[CrossRef][Medline]
42. Rode, L. M., W. Z. Yanf, and K. A. Beauchemin. 1999. Fibrolytic enzyme supplements for dairy cows in early lactation. J.
Dairy Sci. 82:2121-2126.
43. Rojas, M., and P. L. Conway. 2001. A dot blot assay for adhesive components relative to probiotics in microbial growth in
biofilms. Methods Enzymol. 336:389-402.[Medline]
44. Roos, S., and H. Jonsson. 2002. A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to
mucus components. Microbiology 148:433-442.[Abstract/Free Full Text]
45. Savage, D. C. 1987. Microorganisms associated with epithelial surfaces and the stability of the indigenous
gastrointestinal microflora. Die Nahrung. 5-6:383.
46. Scheirlinck, T., J. Mahillion, H. Joos, P. Dhaese, and F. Michiels. 1990. Integration and expression of -amylase and
endoglucanase genes in the Lactobacillus plantarum chromosome. Appl. Environ. Microbiol. 55:2130-2137.
47. Selinger, L. B., C. W. Forsberg, and K.-J. Cheng. 1996. The rumen: a unique source of enzymes for enhancing livestock
production. Anaerobe 2:263-284.
48. Shils ME, Olson JA, Shike M, eds. Modern Nutrition in Health and Disease . 9th ed. Philadelphia, Pa.: Lea and Febiger;
1999
49. Suarez F, Levitt MD, Adshead J, Barkin JS. Pancreatic supplements reduce symptomatic response of healthy subjects to
a high fat meal. Dig Dis Sci . 1999;44(7):1317-1321.
50. Treacher, R. J. and C. W. Hunt. 1996. Recent developments in feed enzymes for ruminants. Proc. Pacific Northwest
Nutrition Conference. Seattle, WA.
51. Tricarico, J. M., and K. A. Dawson. 1999. Effects of defined xylanase and cellulase enzyme preparations on digestive
processes of ruminal microbial cultures. J. Dairy Sci. 77(Suppl. 1):252. (Abstract)
52. Vandevoorde, L., H. Christianens, and W. Verstraete. 1991. In vitro appraisal of the probiotic value of intestinal
lactobacilli. World. J. Microbiol. In addition, Biotechnol. 7:587-592.
53. Varel,V.H., K. K. Kreikemeier, H.J.G. Jung, and R. D. Hatfield. 1993. In vitro stimulation of forage fiber degradation by
ruminal microorganisms with Aspergillus oryzae fermentation extract. Appl. Environ. Microbiol. 59:3171-3176.
54. William V Dashek (1997). Methods in Plant Biochemistry and Molecular Biology. CRC Press. ISBN 0-8493-9480-5. p.
313 Google Print reference "Xylans can by hydrolyzed by beta-xylanase"
55. Yang, W. Z., K. A. Beauchemin, and L. M. Rode. 1999. Effects of an enzyme feed additive on extent of digestion and
milk production of lactating dairy cows. J. Dairy Sci. 82:391-403.
56. Yanovski SZ, Yanovski JA. Obesity. N Engl J Med . 2002; 346:591-602.