Anderson - New Feed Enzyme Developement

September 2009, ChemGen Corp.

NEW FEED ENZYME DEVELOPMENT
David M. Anderson and Humg‐Yu Hsiao
ChemGen Corp.
ABSTRACT
The feed enzyme Hemicell® containing β‐mannanase provides benefits applied to corn‐soybean feeds
beyond the growth performance improvement that might be expected from a digestive enzyme. These
benefits include improvement of weight uniformity, beneficial use in antibiotic‐free diets, and very
significant improvements for birds under the stress of infections. A theory that ingested
galactomannan, the substrate for β‐mannanase, stimulates an innate immune response was tested.
Evidence that galactomannan behaves as a PAMP for the innate immune system was found through
examination of the effect of Hemicell® addition to feed on the serum levels of acute phase protein α1‐
acid glycoprotein. Using the α1‐acid glycoprotein levels as a screening tool, new candidate feed
enzyme/substrate combinations were identified. New enzymes are very analogous to the β‐mannanase
prototype in terms of known details about the biology of the substrate’s involvement in stimulation of
the innate immune system, and in the ability to improve growth performance. One new feed enzyme, a
novel β‐1,3‐glucanase originating from the same source as Hemicell® β‐mannanase will be described in
detail as an example.
INTRODUCTION
There are only 13 basic categories of enzyme types approved for use in feed by AAFCO that hydrolyze or
oxidize 10 substrate types (1). Although great progress has been made in utilization of feed enzymes
over the last 5‐10 years, to our knowledge, the most widely used feed enzyme formulations only
include one or more of a few enzymes as the major ingredients including: phytase, β‐1,3(4)‐glucanase,
xylanase, amylase, β‐mannanase, and protease. This list has been kept short in part due to regulatory
requirements, but our goal is to expand the list of effective enzymes that can improve meat and egg
production economics.
Identifying novel and effective feed enzymes can be challenging. An enzyme must provide significant
improvement of animal production over systems that are already highly optimized after many years of
research on animal genetics, diets, and improvements in management and feeding practices. The
animal growth tests required to demonstrate success for a new enzyme are difficult requiring a high
degree of replication, excellent attention to detail, and the investment of significant time and capital.
Before animal test can be performed, R&D investment is needed to develop the novel enzyme source.
Essentially, a reasonable expectation of success is needed to justify the R&D investment. For these
reasons we sought to develop new concepts and screening tools to identify feed enzyme candidates.
Here we describe a new feed enzyme and rapid screening methods. It was with experiments designed
to elucidate the mechanism of the feed enzyme β‐mannanase that led to this approach.
D.M. Anderson and H. Hsiao, New Feed Enzymes Page 1

In the early 1990s ChemGen developed a β‐mannanase feed enzyme with the trade name Hemicell®.
After a significant amount of animal testing, as well as many field observations, we concluded that β ‐
mannanase was a novel feed enzyme that did not fit the historical pattern. For example, enzymes like
xylanase and β‐1,3(4)‐glucanase were the first enzymes developed to degrade large amounts of xylan
and mixed link glucan that are in wheat and barley diets respectively . The bulk characteristics of glucan
and xylan present in relatively large amounts interfere with digestion. A second feed enzyme category
includes phytase, amylase, and protease where the goal is to obtain more nutrients out of feed by
improving digestion of materials where there are sub optimal levels or types (e.g. amylase, phytase, and
protease) synthesized by the animal. The β‐mannanase is novel in that the β‐galactomannan substrate
content of a typical corn‐soy diet is relatively small compared to the impact observed by degrading it.
A huge and growing literature base indicates that mannan polymers of various linkages are highly
important biological and reactive polymers. Dated studies, going back to the 1960’s, showed that
feeding galactomannan enriched diets in the form of guar meal had very strong negative effects
increasing feed/gain (2‐9). Ultimately we developed a theory concerning innate immune system (10)
involvement in the β‐mannanase mechanism ‐ High molecular weight mannan, independent of chemical
linkage, appears to be a so called “pathogen associated molecular pattern” or PAMP that activates the
innate immune system through various “pattern recognition receptors” or PRR (11‐13). Importantly,
when mannan is reduced in molecular weight, the PAMP property is lost (12). Through examining the
serum levels of an acute phase protein (APP) called α1‐acid glycoprotein (14), the activation of the
innate immune system by feeding galactomannan, including soy galactomannan, was demonstrated (15‐
17). The β‐mannanase in Hemicell® rapidly reduces the molecular weight of soluble β‐galactomannan
and decreases this immune response. The β‐mannans in clean soybean meal are not pathogen
associated; however in high molecular weight form they must be binding to PRRs to activate a
counterproductive energy‐wasting inflammatory innate immune response. Immune
stimulation/inflammation is known to affect the regulation of growth (18‐22) diverting metabolic energy
away from growth. Also, current theories on gut health predict that gut inflammation alters the
microbiota composition and its normal ability to provide colonization resistance against pathogens (23).
Thus high molecular weight mannans, recognized as a PAMP in the diet, are speculated to have a
negative influence on animal health and growth. Thus immunological effects can fully account for the
large impacts of adding mannanase compared to the amount of mannan in the diet.
Although not anticipated, the ingestion of the PAMP mannan caused significant measurable innate
immue response in chickens and turkeys. The non‐inflammaory secretory IgA (SIgA) production in the
gut is expected to limit the inflammatory response (24, 25, 26), but SIgA is also thought to be capable to
trigger inflammatory responses by facilitating materials transport across the gut wall by the process of
retrotranscytosis. There are several other known factors that impact gut permeability and leakage of
intestinal antigens including maturity level (27, 28), heat stress (29), and neurological factors (30). For
example infection in the avian intestinal wall, (i.e Eimeria species.), no doubt also plays a role in gut
permeability.
Mannan is only one of many PAMPs, so other analogous substrates could exist in feed, either pathogen
associated or not, that may also be destroyed by enzymes added to feed. Examination of acute phase

proteins (APP) in blood provides a relatively quick screen to identify other potentially useful enzyme‐
substrate combinations. APP levels are generally easy to measure since they amplify (or decrease)
rapidly upon innate immune system stimulation (14).
In this review, example data is presented showing the impact of β‐mannanase on serum AGP levels, and
the inverse relationship between serum AGP and growth performance. The rational for a new feed
enzyme we have developed is described and how it was initially validated as a good candidate by serum
acute phase protein screening. The new enzyme’s properties are highly analogous to the qualities of β‐
mannanase as a feed enzyme. Both degrade PAMPs in feed that are recognized by hosts PRRs, and both
enzymes we believe increase feeding efficiency primarily through the elimination of a counterproductive
innate immune response. This novel enzyme for feed has the potential to become another widely
accepted feed enzyme supplied by ChemGen.
ABBREVIATIONS
TERM MEANING
AAFCO American Association of Feed Control Officials
AGP α1‐acid glycoprotein
amylase α‐amylase (EC 3.2.1.1); pullulanase (EC 3.2.1.41);
glucoamylase (EC 3.2.1.3); β‐amylase (EC 3.2.1.2)
ANTS 8‐anino‐1,3,6,‐naphthalene trisulfonic acid
APP acute phase protein
BDG Brewer’s dry grains
BMD bacitracin methylene disalicylate
cfu colony forming units
ChemGen MU million ChemGen units = 4000 IU
ChemGen Unit (U) 250 International enzyme units (IU)
CMP Carboxymethyl pachyman
CP Crude protein
CRD carbohydrate binding domains
DC‐SIGN dendritic cell intercellular adhesion molecule‐3‐grabbing non‐integrin
DDGS Distillers dried grain and solubles
dp degree of polymerization (as in the # of sugars in a sugar polymer)
FC (or FCR) feed conversion (feed injested/weight gain)
GPI‐linked glycosylphosphatidylinositol linked
Hemicellulase enzyme that hydrolyzes a hemicellulose, but historically, a name
particularly applied to β‐mannanase
Hemicellulose any of several non‐celluose carbohydrate polymer components of plant
cells
IGF‐I insulin like growth factor I
IGF‐II insulin like growth factor II
IU International Enzyme Unit ( 1.0 micromole/minute enzyme reaction)
MOS mannose oligosaccharides
MR mannose receptor
NEFA nonesterified fatty acids
PAMP Pathogen Associated Molecular Pattern

PRR Pattern Recognition Receptor
SDS PAGE sodium dodecylsulfate polyacrylamide gel
SPR Soutnern Poultry Research, Athens, Georgia
TLR Toll like receptor
WAFC weight adjusted feed conversion
xylanase endo‐1,4‐ β‐xylanase (EC 3.2.1.8)
β –mannanase endo‐‐1,4‐ β‐D‐mannanase (EC 3.2.1.78)
β‐1,3(4)‐glucanase endo‐1,3(4)‐ β‐D‐glucanase (EC 3.2.1.6)
β‐1,3‐glucanase endo‐1,3‐β‐D‐glucanase (EC 3.2.1.39)

MATERIALS AND METHODS
AGP measurements ‐ Radial Immunodiffusion test plates for chicken α‐1‐acid glycoprotein were
obtained from Cardiotech Services, Inc., 2149 Emerson Avenue, Louisville, KY. Chicken blood was
collected into anticoagulant‐coated tubes, and plasma was prepared through centrifugation to remove
cells. Plasma samples were frozen for shipment and refrigerated until use. Kit directions were
followed, except plates were incubated at room temperature for 2‐3 days before reading, and the
polynomial curve fit function of Microsoft Excel was used to make an equation to calculate unknown
values from diameters. Generally 30 birds were analyzed per experimental treatment. The serum level
APPs (like AGP) can be influenced by several factors including physical injury, thus outliers can be
expected, typically 3‐6% in a treatment group. Values greater than two standard deviations above the
mean value are removed for analysis. AGP measurements less than two standard deviations below the
mean were never observed.
Animal Testing‐ Pen growth trials with chickens or turkeys were conducted at Southern Poultry Research
by Dr. Greg Mathis. Cage trials with chickens were also conducted at the University of Gerorgia by Dr.
Nick Dale, Dr. Adam Davis and Elisabeth Freeman. A swine growth experiment at Purdue University was
conducted by Jon Ferrel and Dr. Brian Richert.
Enzyme Assays‐ endo‐β‐1,4‐Mannanase or endo- β‐1,3‐glucanase assays utilized a reducing sugar
method (31) based on dinitrosalisylic reaction with the enzymatic digests of Locust bean gum (Sigma
Chemical Co.) or carboymethyl pachyman (Megazyme) substrates respectively. An international unit is
defined at 1 µmole of increased reducing sugar ends creation /minute standardized with a pure glucose
or mannose. A ChemGen U = 250 IU or one MU (million ChemGen units) = 4000 IU.
GC analysis of sugars – the total mannose content of feed stuffs as an estimation of possible total
mannan content as previously described (32,33).
Estimation of soluble β‐1,3‐glucan content in feed Ingredients ‐ (A) Removal of oligosaccharides and
lipids by extraction: Samples (15‐20 grams) were ground into fine < 40 mesh, then 10‐15 grams were
suspended into 200ml 80% EtOH in a 500ml flask with a cap. The flask was shaken at 37oC overnight.
Insoluble materials were collected on a 3 mm paper filter and washed with 80% ethanol, 100% ethanol
and hexane consecutively. Finally the powder was dried. (B) Solubilize polymers, precipitate and wash:
Sample extracted in A (300 mg) was placed in a screw cap test tube, 5ml of water was added and
placed in boiling water bath for 30 minutes. After cooling to room temperature, 20ml ethanol was
added and mixed well to re-precipitate high molecular weight polymers. After centrifuging the upper
clear layer of 80% ethanol was removed. Then 7.5 ml of ethanol was added, mixed and solids were
precipitated by centrifugation again. The ethanol was removed, and the extraction repeated with 7.5ml
hexane. The powder was dried. (C) Extraction of water soluble polymers and enzyme reaction: 6 ml
MOPS buffer, pH 6.5 was added into the tube. After mixing well, tubes were placed in a boiling water
bath for 30 minutes, cooled, centrifuged, then 0.3ml was removed from upper aqueous phase into 1.2ml
ethanol to adjust to 80%. Then add 0.3 ml of purified β‐1,3- glucanase ( 20 MU/L ChemGen Enzyme
Units) was added into the remaining MOPS extract solution. The enzyme reaction was incubated at 37

o
C with shaking for 2 hours. The test tube was centrifuged and 0.3 ml of upper layer of the reaction
mixture was added into 1.2ml ethanol like the prior control. (D) Determination of enzyme released
sugar: the 80% ethanol solutions were centrifuged, and the total sugar content in the upper 80%
alcohol phase was measured. The total sugar content after 2 hours of reaction minus the total sugar in
the time zero sample gives the net sugar made soluble in 80% ethanol, thus converted into
oligosaccharides by enzyme hydrolysis.
RESULTS and DISCUSSION
Atypical effects of Hemicell® feed enzyme in early observations
Figure 1 – Uniformity Examples
160 160
Control - 905 Birds, CV = 13.4%
140
Hemicell - 967 birds, CV = 11.3% A 140
Control N = 966 B
120 120 Hemicell N = 958
100
Frequency
100
Freq u en cy
Control Control
80 80
Hemicell Hemicell
60
60
40
40
20
20
0
0
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3
3.1
3.2
3.3
3.4
3.5
3.6
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Body Weight Range Body Weight Range
180 Frequency vs. weight (Kg) diagram with all birds individually
160 weighed. Control population‐ blue line; Hemicell fed populations –
140
Control, n= 697 C red line.
Hemicell®, n= 698
120
(A) Chickens at 49 days pen trial: 905 control birds; 967 birds
F re q u e n c y
100 Control fed Hemicell®, conducted at the PARC Institute operated

80 Hemicell by James McNaughton.
60
40
(B) Ducks at 42 days pen trial: 966 control birds, 985
Hemicell®, work conducted at The Breeding Center of
20
Beijing Duck, Ministry of Agriculture, China.
0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
(C) Turkeys at 21 days pen trial: 697 control birds, 698
Body Weight Ranges Hemicell birds, conducted at Southern Poultry Research.
The first atypical observation during Hemicell use was increased body weight uniformity. Figure 1
provides a few examples of uniformity improvement typically seen in Hemicell® fed poultry. The weight
distribution becomes more uniform with the most noticeable difference being decreased frequency of
low weight animals. These were some early observations, but many more examples are available (34).
The second observation, shown in Figure 2 below, shows the effect of enzymatic digestion of mannan in
feed with Hemicell® when birds are under the stress of necrotic enteritis (35‐37). The most interesting
observation is that as the infection becomes increasingly severe, the percent improvement for
Septembeer 2009, ChemGen Corp.

Hemicell®
® addition commpared to thee control incrreases from 0
0, to 6.1 to 21.9% for Livab
bility and 8, to
o 11
and to 21% for FCR forr the light, mo
oderate and ssevere infections respectivvely.
Figure 2 –– Hemicell® Effects with Exxperimental Necrotic Ente
eritis
Comp pilation of data from three 21 1 day cage
100 1
100 trials w
with chickens infected with
experrimental necrottic enteritis using the
90
A 9
90
diseasse model conditions develop ped by Greg
80 8
80 Mathiis at southern Poultry Researrch at
70 7
70
three levels of severrity. Birds werre infected
C ontrol with EE. maxima, E. A Acervulina and C.
H emicell
60 6
60 perfrinngen as previo ously describedd (35,36).
M edicated
50 5
50 Treatm ments included d (a) control w
with no
feed aadditive, (b) H
Hemicell® at 1000 MU/ton
40 4
40
and (cc) medicated w with Salinomycin at 60
Severe Mediu
um Light
g/ton and BMD at 50 g/ton.
Panel A‐ Livability (%
% survival) of b
birds at
three the levels of innfection intenssity plotted
with ccontrol birds (rred), Hemicell birds
(greenn) and medicatted (blue). Numerical
2.5 2.5
valuess are listed below with statistic
Control
Hemicell
B analyssis. Numbers iin columns witth different
Medicated letters are significan
nt (p<0.05).
2 2
FCR
Panel B – Feed Convversion of birdss at three
the levels of infectioon intensity plootted with
1.5 1.5
contro ol birds (red), H
Hemicell birds (green)
and mmedicated (bluee). Numerical vvalues are
listed below with staatistic analysis. Numbers
1 1
in coluumns with diffferent letters aare
Severe Medium
m Ligh
ht
signifiicant (p<0.05). In red, the reelative % of
the feeed conversion n values in eachh column
compared to the meedicated case ((set at
100%) is calculated for easy comparison.
Hemicell® ® contains no antibiotics annd it does nott improve thee bird’s performance as much as the
medicated d treatments. However, the mannanasse addition prrovides a big improvement over the
infection case without treatment. IIn Table 1 thee kcal of feed used per kg o of body weighht under each
h
level of in
nfection was ccalculated. Th
his is anotherr way to repreesent how the impact of u using mannan nase
becomes larger as the severity of in n trials, Hemicell®
nfection stress increases. IIn field trials and large pen
provides a me
typically p easurable and d significant in
ncrease in livability (38, 39
9).
Positive im
mpacts of Hemicell® addition were also o seen when eexamining some typical biochemical
parameteers in the bird’s serum in th
his infection m
model (Table 2). This stud
dy was performed in
collaborattion with Dr. John P. McMurtry at USDA A and Southeern Poultry Reesearch. Com
mpared to non
n
D.M. Anderson and H.. Hsiao, New Feed Enzym
mes P
Page 7

infected birds, every blood parameter was significantly reduced in the infected‐nonmedicated birds.
Mannanase use significantly improved every parameter measured (except lactate) in the infected birds
and in many several cases returned the levels to near the uninfected control.
These experiments illustrating improvements in body weight uniformity, improved performance under
high stress of infection, and increases in the insulin like growth factor IGF‐I are impacts of mannanase
that we did not expect (Table 2). At this point a significant effort was made to learn everything known
about the biological effects of mannan and how it could have an influence in animal feed.
Table 1‐ Caloric Conversion in necrotic enteritis infection experiment of Figure 2 (kcal/kg wt.)
Infection Light Medium Severe
Control 4907 5259 7555

Hemicell 4580 4740 6366
Benefit 327 519 1189
Table 2‐ Impact of Hemicell® on blood metabolites1 and some hormones
BMD 50
Hemicell g/ton Uric
100 Salinomycin Lipid NEFA Acid Lactate IGF‐I IGF‐II
Infection MU/ton 60 g/ton (mg/ml) (Ueq/L) (mg/L) (mg/dl) ng/mL ng/mL

‐ ‐ + 260.65a 638.04a 63.37a 46.98b
30.18a 77.55a

+ ‐ ‐ 509.21b 377.64b 53.86b 56.72a 24.58b 42.44c

+ + ‐ 394.34a 633.07a 60.53a 51.89ab 30.17a 67.85b
1
Blood metabolites were analyzed in the laboratory of John McMurtry at the USDA, chickens were grown and
infected with experimental necrotic enteritis at Southern Poultry Research.
Proof that mannanase is the active ingredient in Hemicell®
The commercial product Hemicell® is produced by a non‐GMO natural isolate that produces an extra-

cellular complex mixture of numerous enzymes. It is formulated for mannanase level, but also has
significant levels of β‐1,3(4)‐glucanase (barley type glucanase), amylase and a large number of other
digestive enzymes in lesser amounts. In order to prove that degradation of mannan is essential for the
mechanism in corn‐soybean diets, first data showing that the mannanase is indeed the active ingredient
was important. Figure 3 A shows an SDS PAGE analysis. The middle channel of the gel shows that the β‐
mannanase has been purified to homogeneity. This preparation was used in a feeding trial compared
to no enzyme addition and crude Hemicell®. This test was done to reproduce a much earlier trial
performed in the 1990s. The gel analysis shows that the purified mannanase tested was virtually
homogeneous.

In a 42 daay broiler triall birds growth
h was significantly improveed for both H
Hemicell® and
d homogeneous
mannanase treatment over no enzyyme control, and there waas no significaant differencee between thee two
enzyme trreatments.
Figure 3 – Purified M
Mannanase u
used in broiller growth sstudy
Broiler Trial -42 Days
1.9
a
A 1.87
B
1.84 b
WAFC
b
1.81
1.78
1.75
Mannanase from Hemice ell® was purifieed by chromato ograpy on DEAAE‐Sepharose aand Phenyl‐Sep pharose using

traditional methods. Pan nel A: SDS PAGGE gel comparin ng crude Hemiicell® and puriffied mannanasse. Panel B: Weight
adjusted feeed conversion n vs. treatmentt. Different low
wer case letterrs on bars signify statisticallyy significant
differencess (P < 0.05). Co
onducted at SPPR.
These results indicate that mannanase is the com mponent imp portant for im

mproved grow wth in normal
healthy birds, but the ppure enzyme was also testted in the neccrotic enteritis disease mo odel at Southeern
Poultry Reesearch to bee sure that maannanase is the componen nt necessary ffor the even larger percenntage
improvem ments observe ed vs. infected control . Thhe same lot off purified mannanase wass used. The reesults
in Figure 4
4 demonstratte virtually identical perforrmance for purified mannanase and Heemicell®. Theese
two feeding trials indiccate that man nnanase is thee active ingreedient. More recent evidence comes frrom
work withh a heat resisttant β‐mannaanase. Fermeentation broth with heat reesistant mannanase was
heated att 83 °C to inacctivate all enzzymes except mannanase, and then add ded in pen triials at 100 MUU/ton
mannanase and bird performance w was compared to a control without mannanase and Hemicell®.
Performance between normal and heated heat‐resistant Hem micell® was aggain equivaleent in several tests
(data not shown). It is clear that the mannanasee is the activee ingredient in n corn‐ soybeean diets.
mes P
Page 9

Figure 4 ––Pure mannanase perform
mance in necrrotic enteritiss model
Cage trial ffor 21 days con
nducted at SPR R with the necrrotic enteritis m model. All treatments infectted with E. maxxima,
E. acervulinna, and C. perffringens. Medicated treatmeent contained SSalinomycin 60 0 g/ton and BM
MD 50 g/ton.
Different loower case letteers on bars signify statistical significant diffference (P < 0.0
05). Hemicell® or β‐mannanaase
were addeed at 100 MU/tton +/‐ 5 MU. Purified mannanase is as desscribed in Figure 3.
Hemicell® Feed Enzym
me and Inna
ate Immune Response
Legumess like soybeans contain ggalactomann nan in a porttion of the seeed called th he endosperrm

illustrateed in Figure 5
5. The domeesticated US soybeans haave a very sm mall remainiing endospeerm.
In corn‐soybean dietts the galacto omannan co ontent is derrived essentiially all fromm the soybean
meal (33). The 44% protein soyb bean meals hhave low galactomannan (estimated d average 1.61
%) and evven lower in n 48% proteiin meals made with dehulled beans (estimated average 1.2 26 %)
since mu uch of the enndosperm sticks to the to o the hull . TThe active in
ngredient mannanase on nly
lowers th he molecular weight of ggalactomann nan producin ng primarily mannose oligosaccharid des
as illustraated Figuress 6 and 7. Th
he extent of α‐1,6‐ side cchains on the β‐1,4‐man nnan backbo one
affects thhe mannan’ss solubility and also the degree of diigestion. A h higher modiffication level
but also causes somewh
increasess solubility, b hat higher deegree of polyymerization (dp) for a lim mit
digest. OOn the otherr hand β‐1,4‐‐mannans w with a low galactose mod dification have low solubbility
in water,, and water ssolubility is n
necessary fo
or efficient β
β‐mannanasse action. The
galactom mannan digesstion in Figure 7 uses higghy soluble Locust bean gum galactomannan th hat
has a simmilar high deggree of α‐1,66‐ galactose side chain ccontent as so oy galactom mannan. It caan be
seen thatt MOS with dp ranging ffrom 2 to weell over 10 iss produced. N No fragmentts are seen
without m mannanase addition.
mes Page 10

Figure 5 – Endosperm and galactomannan in Legumes
en
Photographs provided by Dr. James A. Lackey. Seed types are (a) Kudzu; (b) wild soybean; (c) US domesticated
soybean; (d) Guar. The endosperm composed largely of galactomannan is labeled “en”. The cotyledon or embryo
is rich in protein and is increased in size in domesticated soybean.
Figure 6‐Hydrolysis of Legume galactomannan by endo‐1,4‐β‐D‐mannanase (EC 3.2.1.78) of Hemicell®
Legume galactomannan
α-1,6-galactose
β-1,4-mannose
n
Hydrolysis Sites
Figure 7 – Oligosaccarides produced from Locust Bean gum galactomannan
1 2 3 4 5 6 Acrylamide electrophoresis of oligosaccharides deriviatized with
fluorescent ANTS to create negatively charged molecules that
dp 10 are separated by size (40). Gel composed of 28% acrylamide
and 2% bisacrylamide was used and was photographed under
dp 4 UV light. Lane 1 – maltodextrin with degree of polymerization
(dp) of 4‐10; Lane 2‐ maltodextrin dp 2‐4; lane 3 – glucose ;
Lane 4 – Locust bean gum; Lane 5 and 6– Locust bean gum
dp 2 galactomannan digested by Hemicell® mannanase. Glucose runs
in the dye front including non reacted ANTS.

The mechanism for β‐mannanase function requires only the reduction of the molecular weight.
The enzyme α‐galactosidase is co‐expressed in most microbial strains that make, β‐mannanase
and this enzyme could create more extensive breakdown of galactomannan if the α‐
galactosidase is a type capable of reacting with MOS. The data here shows that a further
degradation of soy galactomannan into monosaccharides is not necessary since purified β‐
mannanase alone provides the effect.
Mannan and the innate immune system
Mannans of various types are recognized PAMPs by the innate immune system by several PRR
including the serum protein mannose binding lectin (MBL) as well as several cell surface
receptors including mannose receptor (MR) and others (DC‐SIGN) on key immune system cells.
Toll Like receptors (TLR2, TLR4, TLR6) are involved in signal transduction after mannan binding
(10‐13). The association of mannan with the surface of numerous pathogens has likely led to
mannan’s conserved recognition by the innate immune system. The MBL is particularly
interesting since humans with defective variants were found to have a high frequency of certain
infections (64). Recently MBL has been recognized to be involved in the phagocytosis of
mannose containing pathogens and recycle of necrotic cells that effectively aligns cell surface
TLR binding for signal transduction (12).
As illustrated in Figure 8, MBL associates into structures that have multiple CRD (carbohydrade
binding domains). Each individual CRD does not bind mannose very tightly, but a high
molecular weight mannose polymer on a pathogen surface binds tightly and activates a
response. Higher concentrations of mannose or mannose oligosaccharides can in fact inhibit
binding to mannan specific PRR binding as has been shown various in vitro experiments for
example with HIV and Influenza(41‐42).
Figure 8 – Multi‐CRD binding site structure of MBL

This high molecular weight requirement for binding and activation that is a recurring theme for PRR in
general fit the pattern of observations for mannanase being the active ingredient of Hemicell®, and
suggest that galactomannan from legumes could be a PAMP based on the animal data. There was some
literature that botanical sources of galactomannan can be immunologically active since modified
galactomannan containing extracts from edible fungus and Aloe Vera plants were shown to have
immune stimulation properties (43‐45).
In chicken and turkey experiments, it was convenient to test for innate immune system stimulation by
measuring the level of the APP (46, 47) α1‐acid glycoprotein (AGP) as it correlates with innate immune
response (48‐50). For this we used a radial immune‐diffusion kit as described in the Materials and
Methods. Example results for both chickens and turkeys are shown in Table 3 and 4. In Table 3, a 42
day broiler pen trial showed significant improvement in feed conversion, and also significant reduction
in AGP. The Hemicell® β‐mannanase was essentially able to provide the same effect as the addition of
the growth promoting antibiotic BMD into the diet, although the mechanism is clearly different as there
are no antibiotics in the Hemicell® product.
Table 3: Chicken Broiler Trial 42 Days
Days 0-21 21-35 35-42

ME 2960 3020 3080
CP 22 19.4 17.5
Lys 1.2 1.03 0.9
WAFC Stat. AGP Stat.

control 1.820 a 304 a
BMD 1.772 b 246 b
mannanase 1.756 b 225 b
Cobb X Cobb, 50 birds/pen, 10 cage per treatment, 0.93 sq. ft./bird, 50 g/ton BMD (when used), 60 g/ton
Salinomycin , water and feed ad libitum , 42 days trial at SPR. Dietary information for the calculate metabolic
energy and crude protein for each period are shown. The treatments were (a) control diet (without BMD antibiotic
or mannanase), (b) BMD added but no mannanase, and (c) mannanase added (without BMD). Chicken AGP was
determined by immunodiffusion as described in the Materials and Methods section.
Table 4: 140 Day Tom pen trial
kg AGP
WAFC Stat Wt. gain mg/L Stat
Control 2.592 a 17.413 260.5 a

Hemicell® 2.491 b 18.059 225 b
Hybrid (converter) Tom turkeys, 0‐140 days, 11 birds/pen, ~1.1 ft2 during brooding; ~4.2 ft2 during grow‐out, 88
birds/ treatment, 8 repitions. At the end of the trial blood was drawn for AGP measurement. Dr. Jim Mitchell
provided feed formulations to SPR. The basal turkey feeds were manufactured by the University of Georgia,
Athens, GA.
The pen trial results show AGP was significantly reduced in both chicken and turkey trials, and the
statistical significance of the AGP reduction was equivalent to the feed conversion. This is despite the
fact that many fewer birds were bled for AGP testing than the total number used in the trial. The data is

consistent with the ideea that galacttomannan is bbeing recogniized as a PAM MP by at least some of the PRR
that detecct microbial m
mannan and ccause inflamm mation. It is interesting to
o note that BM
MD as growth h
promotingg antibiotic has been prop posed to havee a mechanism m based on immproving gut health to red duce
gut permeeability and subsequent reelease of inflaammation‐pro oducing toxinns past the gu
ut wall (61). LLike
mannanase, the propo osed mechaniism is related to inflammation reductio on.
Quite connclusive data regarding thee PAMP status of Legume gglactomanan was obtained d in a series o
of
experiments conducted at the Univversity of Geo oduction was abbreviated to
orgia. The tesst for AGP pro
weeks startingg with day old
just two w d chicks (15‐117). A basal d
diet was form
mulated with h
highly refined
d
components to be a “lo ow stimulatioon” diet for th
he innate imm mune system to which testt materials were
n these studie
added. In es we observeed that:
(a) Thhe AGP respoonse increased in proportio on to the amount of soybeean meal add ded

(b) A purified Legu
ume galactom mannan, Guarr gum, also sttimulated AGP production
(c) Addition of Heemicell®, or pu
ure mannanaase, reduced tthe AGP levells
(d) AGP reduced in a dose resp ponsive way to increasing Hemicell® maannanase add dition
(e) Exxperiments w
with purified ssoy fractions (soy concentrrate and soy isolate) show
wed that the
reeduced AGP rresponse com mpared to soyybean meal, aand the AGP rresponse was proportional to
th
he mannan co ontent (based d on mannosee detected byy GC analysis of total sugaar).
Some of tthis data is summarized in Figure 10. Baased on all th hese observattions we concclude the soy
mannan iss a PAMP anaalogue. The inflammation it causes can n explain the vvarious beneffits of Hemiceell®
use. The level of 0.4‐00.5% mannan in a typical co much mannan were
orn soy diet is small; howeever, if this m
in pathogen associated d form, it wou
uld be the equivalent of an n extremely laarge dose in tterms of
infectiouss units / kg feed. For exam
mple, if it was a bacteria paathogen (assu
uming a size ssimilar to E. co
oli)
13
with 2% ccell mannan content, abou ut 2.4 x10 cfu /kg could b be estimated.

Figure 10‐‐ Linear corre
elation betwe
een circulatin
ng AGP and fe
eed and soyb
bean derived mannose con
ntent
Chicken ggrowth (1‐14 daays)
and bloodd sampling wass
conducted at the University
of Georgia (15‐17). Bloo od
plasma from 30 birds (3 3 X 10
birds/cage) was analyzeed at
ChemGen n for AGP.
Mannose content (high MW
fraction o
only) of startingg feed
was deterrmined by acid d
hydrolysiss and GC (32,33).
Blue line, no enzyme ad dded.
Red line, 100 MU/ton
Hemicell®® mannanase aadded
mes Page 14

Application of AGP assays to development of new feed enzymes.
If the interpretation of the data is correct there are several implications. An innate response to
food/feed components can have a big effect on inflammation and feeding efficiency. Secondly, because
there are many PAMPs recognized by the innate immune system, there should be other opportunities
for new and useful feed enzymes.
Two approaches using the rapid AGP screening two‐week test are being used. The first is to take feed
ingredients one by one and test if they stimulate AGP production. If stimulation is found, the
component can be broken down to find the PAMP/analogue then try to find an enzyme to break it
down. Figure 10 is one example of this. The increase in AGP by adding soy meal is not reversed 100% by
the addition of β‐mannanase suggesting the soy meal may contain another PAMP. The second approach
is to use what is known about the innate immune system and predict substrate/enzyme combinations
that should be useful to reduce gut inflammation.
A demonstrative example of this second approach follows.
Rational for development of β‐1,3‐glucanase as a feed enzyme
One of the first enzymes targeted for testing was β‐1,3‐glucanase (EC 3.2.1.39). This enzyme should not
be confused with β‐1,3(4)‐glucanase (EC 3.2.1.6) that has been used in feeds for many years. The β‐1,3‐
glucanase has some limited activity on mixed linked glucan substrates form sources like barley and oats
due to occasional blocks of adjacent β‐1,3‐glucose linkages in the polymer, but β‐1,3(4)‐glucanase has
zero activity on pure β‐1,3‐glucan because it only cuts β‐1,4‐glucose bonds as in cellulose. These bonds
do not exist in β‐1,3‐glucan.
β‐1,3‐glucan is a PAMP universally recognized by innate immune systems (51,52) . A PRR for β‐1,3‐
glucan binding called Dectin‐1 is a C‐type lectin highly expressed on dentritic cells, as well as
macrophages, monocytes and neutrophils. Dectin‐1 has been extremely well characterized (51‐55).
Other β‐1,3‐glucan PRR receptors include complement factor 3 (CR3), lactosylceramide, CD5 (56 ) and
more.
The Dectin‐1 PRR is analogous to the mannose receptor (MR) expressed on immune cell surfaces. There
are similarities in properties as well. In vitro studies showed high molecular weight β‐1,3‐glucans such a
yeast cell wall or the soluble high molecular weight bacterial polymer scleroglucan stimulated cytokine
release, but low molecular weight laminarin is antagonistic (51). Several low molecular weight
oligocaccharides with β‐1,3‐linkages have much lower binding constants (53,54) and α‐glucosides do not
bind (55). Importantly, the branched cell wall glucan of Scaharromyces cereviae binds very tightly.
In order for the β‐1,3‐glucanase to be a useful feed enzyme like β‐mannanase, the substrate must be a
PAMP, as it clearly is, but also the substrate must be present at a level significant in feeds. Like
mannan, this is also the case for β‐1,3‐glucan as it is a common structural/storage polysaccharide in
algae, fungi cell walls (including yeast), and higher plants (56‐59). In higher plants β‐1,3‐glucan is called
callose and is readily visible in microscopic thin sections where it can be made brightly fluorescent by

staining with aniline blue fluorochrome. It is found normally associated with plasmodesmatal canals,
abscission zones and on sieve plates in dormant phloem. Interestingly it is also synthesized and
deposited between the plasma membrane and the cell wall as a rapid response to range of stresses
including wounding, desiccation, metal toxicity, and microbial attack (58). Callose has also been found
to act as a molecular sieve creating semi‐permeability of a seed endosperm envelope (59). Pollen tubes
and pollen coats are composed of β‐1,3‐glucan. In light of the PAMP status, it is not surprising that
many people have adverse reactions to pollen.
At this moment we have not determined the range of β‐1,3‐glucan content in the most commonly used
feed ingredients, and in particular how the content varies with the crop’s growing conditions. However,
there are feed ingredients derived from yeast alcohol fermentations with significant use, DDGS, BDG
(brewers dry grains, Asia) and yeast by‐products from sugarcane ethanol production in Brazil that
should have relatively high β‐1,3‐glucan content since almost 50% of the dry weight of yeast cell walls is
β‐1,3‐glucan.
It was reported in swine experiments that as little as 100 ppm of extracted yeast glucan in feed reduced
ADG average daily gain (62). Also, with some lots of DDGS, we have observed a strong stimulation of
plasms AGP production compared to similar control diets without DDGS addition. Therefore, we wanted
to obtain a good estimate of the yeast content and associated β‐1,3 glucan in DDGS. A picture of the
organization of β‐1,3‐glucan in yeast cell wall is shown in Figure 11.
Simplified representation of yeast cell wall
showing components (63). The β‐1,3‐glucan
Figure 11: Representation of the polymers is yeast cell wall
(green) forms the backbone of yeast cell walls
and is cross‐linked with chitin (light blue).
Long side chains of β‐1,6‐glucan (red) are
attached periodically to the β‐1,3‐glucan.
Attached to the β‐1,6‐glucan branches are
glycoproteins (blue) extensively glycosylated
with α‐mannans (yellow) that can be up to 95
% of the glycoprotein weight. Considering
whole dry yeast, 8‐9% of total weight can be
mannan. Mannoproteins are initially
synthesized as GPI‐linked membrane bound
proteins. In S. cerevisiae the β‐1,3‐glucan
represents 50% of the wall mass, β‐1,6‐
glucan represents 10% and manno‐proteins
about 40%.
Fifteen DDGS samples were collected from 5 suppliers from three production plants per supplier, or
three seasonal times if a supplier had an individual production plant. These samples as well as brewers
dried yeast were analyzed for the content of β‐1,3‐glucan using a combination of total sugar analysis
(excluding cellulose) and digestion of extracts with purified β‐1,3‐glucanase.

First the purified β‐1,3‐glucanase was characterized to establish the purity and specificity of the enzyme.
The purified enzyme was not 100% pure, but highly purified and free of other glucanases (amylase and
cellulase) . The purity is analyzed in Figure 12 and specificity in Figure 13.
Figure 12 ‐ Purified β‐1,3‐glucanase on SDS‐PAGE gel
Purified β‐1,3‐glucanase from Hemicell®
production host strain analyzed on SDS
page gel (Lanes 2 and 3) compared to
Bio‐Rad standard broad range protein
molecular weight markers (Lane 1).
Marked bands on the left are molecular
weights in kilo Daltons.
Lane 2, normal loading, Lane 3, heavy
loading to check purity. The β‐1,3‐
glucanase is marked with an arrow.
Figure 13‐ Verification of the specificity of purified β‐1,3‐glucanase with defined β‐1,3‐glucans, CMP
and Scleroglucan Carbohydrate gel as described in Figure 7
modified with higher % gel. CMP and was
obtained from Megazyme and Scleroglucan
from Cargill.
Carbohydrate Gel lanes
1‐glucose (dp 1)
2‐ maltose (dp 2)
3‐maltotriose( dp 3)
4‐ maltodextrin (dp 4‐10)
(dp 8‐10 not resolved )
5‐ CMP (contaminated with glucose, no
enzyme)
6,8 –CMP (carboxymethyl pachyman)
digest, two levels loading
7,9 – scleroglucan digest, two levels
loading
32% acrylamine gel, 2 hours, 200 V
Lanes 6‐9 digested with
β‐1,3‐glucanase before ANTS reaction
The purified β‐1,3‐glucanase was able to digest two known available polymers with β‐1,3‐glucan
backbone, carboxymethy pachyman and sclerogucan. Further the gels show that that the enzyme had

endo cutting ability sin
nce oligosacchharides with aa dp in the range of 2‐7 aree visible on th
he gel, but un
ncut
not display an
CPM did n ny of these fraagments.
The mann nan and β‐1,3 3‐glucan analyysis summaryy for DDGS samples are sho own in Table 5 as analyzed

d as
describedd in the Materrials and Methods section. A mannose content (totaal: 1.5%; soluble: 0.9%) in
DDGS wass measured (ssee Figures 14 4 and 15 for iindividual suggar data) and β‐1,3‐glucan
n content of 1
1.2‐
1.5 % is predicted. Meeasured β‐1,33‐glucan by enzyme digesttion in the soluble fractionn is comparedd to
mannan d data in Figure
e 16.
Table 5 – Summary of mannan and
d β‐1,3‐glucan
n content (%)) in DDGS sam
mples
% Corrn DDGGS Yeast

Oil 3.3
3 10
CP 7.5
5 26
6‐28 40
mannan 0.0
05 1.2
2‐1.5 8‐9
β‐1,3‐gluccan 0 1.2
2‐1.5 8‐9
Figure 14 GS samples by GC
4 ‐ Sugar anaalysis of DDG
Top Panel – Mannosse Data
Bottom Panel – Gluccose Data

Data iis shown as aveerage of
three DDGS sampless from each
company. Blue bars show the
sugar polymer conteent that
could be extracted rreadily with
waterr. The Red bars show the
sugar that could be hydrolyzed
by aciid (note, not sttrong acid so
celluloose is excludedd).
Some DDGS samples have a lot
of leftt over starch based on the
glucosse analysis.
mes Page 18

Figure 15
5: Analysis o
of xylose suggar content of DDGS sam
mples
1
10.0 A highh xylose conteent is
expeccted in all DDG
GS samples
8.0
due too concentrating the corn
6.0 xylan by three timees. However,,
% DW its alm
most all poorlyy soluble.
4.0 Soluble
Total Xylosee
2.0
0.0
#1‐3 #4‐6 #7‐9 #10‐12 #13‐15
GS
Source of DDG
Figure 16 – Determinaation of availaable β‐1,3‐glu
ucan in indiviidual DDGS samples by GC
C and purified
d β‐
1,3‐glucan
nase digestio
on (see Materrials and Metthods).
The ethan nol fermentattion process rremoves the sstarch from the corn addin ng solubles annd whole yeaast

into the D
DDGS by‐prod duct. So in Taable 5 corn an
nd yeast are ccompared to tthe DDGS. A About three to ons of
corn results in one ton n DDGS. The oil content inn DDGS is thu us about threee times the oiil level in corn
n
unless it iss extracted out. Mannosee content (tottal: 1.5%; solu uble: 0.9%) in DDGS mainlyy comes from m
added yeaast since the mannan conttent in corn iss only 0.05%. Since 8‐9% d dry wt. of yeaast is mannann,
DDGS con ntains about 1 12‐14% yeast. The Crude Protein (CP) in DDGS is 4‐5 % points higher than thrree
times the corn protein factor. This extra protein n is from yeasst since it contains 40% CP. The observved β‐
1,3‐glucan n content in tthe soluble fraction was generally loweer than the ssoluble mannan fraction. Both
must alsoo be derived from the yeasst content. Th he relatively hhigh yeast conntent in DDGS means thatt in
high DDGS diets, the available β‐1,3 3 glucan conteent in the dieet can be similar to the galactomannan n
levels enccountered in ssoy diets, and d much higheer than the 10 00 ppm wheree negative efffects were
observed in ADG in sw wine (62).
mes Page 19

The high molecular weight sugar polymer extracted from DDGS was compared to a similar extract from
brewers dry yeast after digestion with purified β‐1,3‐glucanase in Figure 17. The fragmentation pattern
is similar to the yeast extract as would be expected. Soy hull extract hydrolyzed with the purified β‐1,3‐
glucanase has a slightly different pattern without a dp 5 fragment. An extract from soy concentrate, a
protein enriched had much reduced glucanase substrate.
Figure 17 – Carbohydrate gel analysis of the fragmentation of soy hull, DDGS and dried yeast water
extracts with purified β‐1,3‐glucanase
Carbohydrate gel as described in
Figure 7 modified with higher %
gel.

Gel Lanes
1‐glucose
2‐ maltose
3‐maltotriose
4‐ maltodextrin dp 4‐10
(dp 7‐10 not resolved)
5‐ soy concentrate extract
6 –soy hull extract
7,8 – DDGS extract
9 ‐dried brewer’s yeast extract

32% acrylamine gel, 2 hours, 200 V
Lanes 5‐9 digested with purified
β‐1,3‐glucanase
More work is needed to understand the β‐1,3‐glucan content in feed components to decide the
best applications of the new enzyme. Products with high yeast content are the area of current
focus. Table 6 shows the glucan content from several additional raw materials including the
DDGS
It is very clear that the β‐1,3‐glucan is a strong PAMP and that it is found in feed from many
possible sources. Animal testing data provided below shows that a broiler 21 day chicken cage
screening test lowered AGP similar to the observation with mannanase. This was followed by
larger growth tests showing that the addition of ChemGen’s novel source of β‐1,3‐glucanase
can, in some cases, significantly improve the growth performance benefit of Hemicell®. The
degree of the added benefit has ranged from very large to moderate, and the current theory is
that the difference likely relates to the β‐1,3‐glucan content in feed that was not defined in
initial experiments.

Table 6 – β‐1,3‐glucan content of DDGS and yeast feed products
% DDGS DDGS Brewers BDG BDG BDG MDG

Avg. 15 USA Dry China-1 China-2 Philippines China
samples midwest Yeast
Total 0.97 0.843 6.17
mannose
Soluble 0.561 0.322 2.009
mannose
Total 5.918 7.873 6.179
glucose
Soluble 2.512 4.62 1.334
glucose
β‐1,3‐ 0.298 0.525 3.814 1.02 0.79 0.5 1.012
Glucan
Testing β‐1,3‐glucanase in Feeding Experiments
The enzyme β‐1,3‐glucanase actually already exists in Hemicell®, but at a low level. The β‐1,3‐glucanase
expression level in the Hemicell production strain was increased to a commercially useful level to
provide a safe supply from a host that does not contain any foreign DNA sequences. This feed‐approved
microbial strain also naturally produces a number of other potentially useful enzymes for feed that we
plan to exploit. The new strain produces β‐mannanase and the β‐1,3‐glucanase simultaneously. The
target dose for the β‐1,3‐glucanase like Hemicell® mannanase has been 70 to 100 MU/ton (ChemGen
Units).
The new enzyme will be sold in combination with mannanase, but in some trials it was tested separately
in a more purified form. Initially a 21 day cage trial was performed at Southern Poultry Research as
described in Table 7. The WAFC was significantly improved in the short 21 day test, and AGP was
significantly reduced over control. This early result indicated that further testing and development of β‐
1,3‐glucanse as a feed enzyme was warranted.
In the first pen trial shown below was a 42 day turkey hen experiment comparing β‐1,3‐glucanse to a
control, and Hemicell® and β‐1,3‐glucanse plus Hemicell®. This trial used a commercial type corn‐soy
diet and feed was produced at a commercial mill. The results in Table 8 showed that this form of β‐1,3‐
glucanase significantly reduced the AGP (again looking at AGP in a sub population of 30 birds only) but
adding the combination of mannanase and glucanase did not reduce AGP further. The β‐1,3‐glucanase
also significantly improved the feed conversion over the control. Unlike the AGP result, the growth
performance was significantly better when the enzymes were combined. The Hemicell® and β-1,3-
glucanase + Hemicell® data is the average of two separate treatments in each case for a very high
degree of replication.

Table 7‐ Chicken 21 day broiler trial at SPR with β‐1,3‐glucanase to test AGP response
Stat. AGP Stat.

Treatment WAFC P=0.05 mg/L P=0.05
control 1.474 a 255.4 a
Hemicell 1.390 ab 184.1 b
1,3- glucanase 1.264 c 157.7 b
One‐day‐old male Cobb X Cobb chicks were obtained from Cobb‐Vantress hatchery, Cleveland, GA on April 20,
2006. Chicks were raised in Petersime battery cages. Space per animal was 0.631 sq.ft/bird (588 sq.cm/bird), 8
birds per cage. The feeder space per bird was 8 birds/43 cm x 6.8 cm feeder. Thermostatically controlled gas
furnace/air conditioner maintained uniform temperature. Even illumination was provided. Cages were blocked by
location in the battery with block size equal to treatments (21 cages per block and 3 replications per treatment).
Feed and water were given ad libitum. On Day 21, approximately 3 mls of blood were collected from each bird.
Blood was placed into a EDTA coated tube and lightly mixed. Tubes were slowly centrifuged and then serum was
removed. Serum samples were placed into tubes with caps labeled with cage number. Serum was frozen and sent
to ChemGen for AGP analysis by radial immunodiffusion.
Table 8 ‐ Turkey Hen trial, 42 days
Treatment WAFC Stat AGP Stat

control 1.520 a 214.8 a
Hemicell 1.5021 ab 177.2 ab
β-1,3-glucanase 1.465 c 172.1 b
1
β- 1,3-glucanase + Hemicell® 1.402 d 171.3 b
One‐day‐old female turkey poults were obtained from Tar Heel Hatchery, Raeford, NC. The birds were kept in 80
pens each having an area of 5 x 10 = 50 ft2 , with built up poultry litter topped with fresh pine shaving as bedding for a
thickness of approximately 4 inches. There were 10 pens per treatment and 40 hens/pen. The stocking density, after
subtracting out for equipment, was 1.16 ft2/turkey‐hen. All basal feeds were manufactured at Purina Feed Mills,
Gainesville, GA. Coban, at 90 g/t, was added to the pre‐starter feeds by SPR. Turkey AGP was determined by
immunodiffusion as described in the materials an methods, but the turkey AGP kits have been discontinued. 1Note
– data for Hemicell® teatment and β‐ 1,3‐glucanase + Hemicell® treatment are the average result from duplicated
treatments.
An additional research trial at Southern Poultry Research was recently completed. The basic diet
compositions, crude protein, metabolic energy, and cost for each feeding period are shown in Table 8.
Data from 84 days is plotted in Figures 18‐20. The corn control diet is an optimal corn soy control with
high caloric content. The objective was to see if the novel β‐1,3‐glucanase or Hemicell® could improve
diets containing DDGS. Thus there are two more diets, one with DDGS plus energy equivalent to the
corn/soy diet and a DDGS diet with low caloric content that that was expected to be replaced by enzyme
use. The enzymes were added to the DDGS low energy diet. In general the enzymes are improving the
negative DDGS control up to the level of the DDGS positive control, except at 42 days where the
difference between the DDGS positive control and corn soy control for some reason became quite large.
From these results it appears the addition of β‐1,3‐glucanase did not provide significantly better feed
conversion than the than the β‐mannanase alone in the diets.

Table 8 –Basic diet composition for SPR100708 Turkey hen trial
Corn Pos. Neg.

Control Control Control
Prestarter 0‐3 weeks
Corn 41.9 39.7 40.3
SBM 45.9 43 42.2
Fat 1.2 1.2 0.5
Poultry Meal 6 6 6
DDGS ‐ 5 5.7
CP 29 29 29
ME 2850 2850 2817
Cost ($)/ton 335.18 330.07 325.19
Starter 3‐6 weeks
Corn 43 38.7 40.4
SBM 43.6 37.8 36.1
Fat 2.7 2.7 0.5
Poultry Meal 6 6 6
DDGS ‐ 10 12.3
CP 28 28 28
ME 2950 2950 2850
Cost ($)/ton 338.97 329.14 314.1
Grower 6‐9 weeks
Corn 49.7 43.3 46
SBM 38.3 29.6 26.8
Fat 4.2 4.3 0.5
Poultry Meal 4 4 4
DDGS ‐ 15 18.8
CP 24.5 24.5 24.5
ME 3100 3100 2928
Cost ($)/ton 328.44 313.24 287.94
Finisher 9‐12 weeks
Corn 60.1 53.6 56.8
SBM 28.2 19.6 16.2
Fat 4.8 4.9 0.5
Poultry Meal 4 4 4
DDGS ‐ 15 19.4
CP 20.5 20.5 20.5
ME 3250 3250 3050
Cost ($)/ton 311.61 29.24 266.64


Figure 18‐ SPR100708 TTurkey Hen TTrial Weight G
Gain up to 84
4 days

Figure 19 – SPR100708
8 Turkey Hen
n Trial Feed Co
onversion trial up to 84 days
mes Page 24

Figure 20 ‐ Figure 19 –
– SPR100708 TTurkey Hen TTrial Feed Con
nversion triall up to 84 dayys
Table 9 ‐ TTurkey Hen TTrial Cost perr live pound ccalculation
Cost ($) per
Diet Diet Typ
pe Enzyme live lb
b.
Corn/Soy CControl Corn/soy ‐ 0.312
2
Positive control DDGS + energy ‐ 0.312
2
Negative CControl DDGS + low eenergy ‐ 0.292
2
Negative CControl + Enzyyme DDGS + low eenergy 1,3‐Glucanasee1
β‐1 0.285
5
Negative CControl + Enzyyme DDGS + low eenergy Hemicell 0.288
8
1
the β‐1,3‐‐Glucanase pre
eparation also contains Hemicell® mannanaase
However, taking into aaccount feed cost for the d different dietss and live pou
unds of turkeyy produced; tthe
combination enzyme h has the least ccost (Table 9)). More workk is needed too understand why some triials
with β‐1,3
3‐glucanase wwere very possitive with add ditive enzymee benefits, annd this last turkey trial wass not.
One pointt to keep in m
mind is that th
he yeast in DDDGS is bringin
ng in a lot of α
α‐mannan PA AMP that is noot
being neu
utralized by enzyme digesttion.
An interessting experim
ment was condducted at Purrdue Universiity by Brian Richert,
R Ph.D.. and Jon Ferrrel of
ChemGenn Corp. to dettermine the eeffect of addin ng β ‐mannan nase or β‐ maannanase pluss experimental β‐
1,3‐glucan
nase to a cornn‐soybean meeal‐ DDGS dieet on pig growwth, feed efficciency, and overall
performance during th he grower ph
hase. Pigs (in
nitial BW = 18.0 ± 0.16 kg) were allocateed in a
mes Page 25

randomized complete block design (RCBD) of mixed gender pens, stratified by litter source and initial
weight, into three (3) study treatments. There were ten (10) replicates per treatment, two (2) per
room. Dietary treatments included: Negative Control and the Negative Control diet profile plus enzyme
for treatments (Hemicell® β‐mannanase or Hemicell® β‐mannanase + β‐1,3 ‐glucanase at two levels).
Diet formulations were changed every three weeks with DDGS concentration increasing from 15% to
20% and 25%. Data analysis is still in progress and the data will be published independently. However,
indications from early time points show β‐1,3‐ glucanase providing significant improvement over the no
enzyme and Hemicell® controls. (Jon Ferrel, personal communication).
CONCLUSIONS
The innate immune system has been highly conserved through evolution with analogous elements
found from insects, to plants to humans. Indeed, β‐1,3‐glucans elicit an evolutionarily conserved innate
immune response most likely due to its almost universal inclusion in fungal cell walls. The innate
immune system with its powerful and rapid response is essential for survival due to its role to fight
infections prior to the development of adaptive immunity in animals. However, it is a two edged sword.
Analogous to the problem of the unwanted autoimmune diseases occasionally caused by faulty
regulation of the adaptive immune system, we are learning that the innate immune system reacts in a
counterproductive way against non‐pathogen food components that happen to be analogues of PAMPs.
As more is learned about the immune systems and related scavenger systems, there should be more
opportunities to use enzymes to degrade PAMPs and PAMP analogues that are not actually pathogen‐
associated in order to reduce the inflammation that they cause. We believe that this will further
improve feeding efficiency and animal health. Mannanase and a novel β‐1,3‐glucanase are the first two
examples of several possible new and useful catalysts in animal feed. A third enzyme ChemGen has in
development looks very promising and was selected using the same model. ChemGen believes that
taking advantage of biotechnology to produce useful quantities of new feed enzymes of this category is
a cost effective and efficient way to make improvements in meat and egg production.

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September 2009, ChemGen Corp.

Body Weight Range Body Weight Range

100 Control fed Hemicell®, conducted at the PARC Institute operated

Infection Light Medium Severe

Control 4907 5259 7555

The commercial product Hemicell® is produced by a non‐GMO natural isolate that produces an extra-

Broiler Trial -42 Days

Mannanase from Hemice ell® was purifieed by chromato ograpy on DEAAE‐Sepharose aand Phenyl‐Sep pharose using

These results indicate that mannanase is the com mponent imp portant for im

Legumess like soybeans contain ggalactomann nan in a porttion of the seeed called th he endosperrm

Days 0-21 21-35 35-42

WAFC Stat. AGP Stat.

Control 2.592 a 17.413 260.5 a

(a) Thhe AGP respoonse increased in proportio on to the amount of soybeean meal add ded

The mann nan and β‐1,3 3‐glucan analyysis summaryy for DDGS samples are sho own in Table 5 as analyzed

% Corrn DDGGS Yeast

The ethan nol fermentattion process rremoves the sstarch from the corn addin ng solubles annd whole yeaast

% DDGS DDGS Brewers BDG BDG BDG MDG

Stat. AGP Stat.

Treatment WAFC Stat AGP Stat

Corn Pos. Neg.

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