Note: Descriptions are shown in the official language in which they were submitted.
2131~90
0 93/19162 ~ PCT/US93/00867
Title: FATTY ACID MICROSPHERES CONTAINING
ENTEROCOCCUS FC)R USE TO ENHANCE GROWTH AND
IMPROVE CARCASS QUALITY
BACKGROUND OF THE INVENTION :
Growth enhancers in the form of antibiotics have
been used extensively for poultry, namely chickens and
turkey. Growth enhancers such as Stafac ~ and B
(bacitracin methylene disalicylate) are k~own
antibiotics and have been used at sub-therapeutic
levels of for example, 10 grams per ton and 25 grams
per ton as feed additives in order to promote
~-~ desirable growth features in poultry. However~ the
use of antibiotics for these purposes has recently
come under some criticism. One of the criticisms is
the possibility that the poultry eventually develop `~
tolerance to the antibiotics and eventually the
antibiotic no longer works well for growth promotion.
Other ob~ections relate to health concerns from non-
natural antibiotic additives and the adulterating
effects they may have. Nevertheless, because of the
advantages of antibiotic uses they are still commonly
used in order to improve feed conversion, improve
carcass composition, and enhance growth.
It is known that certain bacteria are potentially
beneficial when added to animal feeds. These bacteria
are beneficial in that they supply a natural
intestinal micro-flora. Some companies offer for sale
direct-fed microbials which contain desirable
bacteria. Direct-fed micr~bials, however, do have
some difficulty in maintaining a stable product.
Typically, the direct-fed microbial is used at a
fairly low level, added to feed at perhaps a O.l~
level. However, unused direct-fed microbial
.,
WO93/1~162 PCT/~S93/008~.
2131~90
-- 2
containing feed or feed additive product is often
stored by the farmers for long periods of ~ime. This
storage many times is under conditions where there is
some moisture and hi~h temperature. In many instances
there is just enough moisture that the bacteria are
activated or start to grow, but yet there is an in-
sufficient amount of moisture to sustain them. As a
result they die. Thus, the activity of the direct-fed
microbial is stopped. In other instances, the
addition of antibiotics to the direct-fed microbial
contalning feed or feed additive adversely interacts
~-'with the bacteria, particularly if there are small
amounts of moisture present and thus again bacteria
are killed. Thus F there is a significant problem of
long term storage stability for direct-fed microbials.
In another environment, where the direct-fed
microbial is added to, for example chicken feed, it is
common to pelletize the material with the direct-fed
microbial added before pelletizing. Moisture from -~
steam used during pelletization partially activates
the bacteria, but may, as a result of insufficient
moisture to sustain them, kill them. Also heat during
pelletization may kill them. Then, too, there is the
problem of the acid environment of the stomach
potentially inactivating bacteria before they really
reach the intestine. Thus, there is a continuin~
need for direct-fed microbials which will release the
organisms only at the proper time in the intestine,
without early release due to moisture conditions or
adverse pH conditions such as exist in the digestive
tract anterior to the small intestine.
`~093/19162 2 1 3 ~ 7 9 0 PCT/~S93J00867
- 3 - -
~ .
Certain features of poultry are especially
desirable to achieve if possible. Those include an
increased rate of weight gain, better feed conversion,
carcass composition, and finally uniformity of flock
weight. Increased rate of weight gain and better feed
conversion are, of c~urse, desirable for the attendant
economics that accompany these desirable results. The ~;~
composition of carcass is important because the most
desirable area for tissue deposit is the breast in
order to yield a high amount of choice meat. Thus,
weight gain is not only important, but where the
weight is gained on the carcass is also important.
Uniformity of flock weight is important because if
more birds are normal in size, less hand labor is -~
required and processors can more extensively rely on
machine processing. On the other hand, if the birds
vary considerably from very small birds to very large
b~rds, even though the overall flock weight may be the
same, the smaller birds and the larger birds require a
great deal more hand labor and because of their lack
of uniformity in size, cannot be processed easily by
machine. Thus, uniformity of flock weight with a high
percentage distribution within the normal size range
so that chickens can process by standardized machinery
is a desirable feature.
Similarly, a direct-fed microbial which is not
only useful for poultry, such as chickens and turkeys,
but also useful for swine would be highly beneficial.
It is a~primary objective o~ the present
invention to provide a poultry direct-fed microbial
which contains no antibiotics and contains only fatty
acid microspheres containing naturally occurring
organi8m8 .
WO93/19162 P~T/US93/008~
2131~790
-- 4
It is another primary objective of the present
invention to provide a direct-fed microbial which
contains two organisms, namely Enterococcus faecium
301, DSM No. DSM-Nr. 4789, and Enterococcus faecium
202, DSM No. DSM-Nr. 4788. ;~SM is a sacterial Culture
collection in Germany. DMS stands for Deutsche
Sammlung von Mikroorganismen located in Braunschweig,
West Germany. The~e organisms will be deposited at
the ATCC, with all restrictions lifted upon notice of
allowable claims.
It is a further objective of the~present
invention to provide a direct-fed microbial which, for
poultry, provides increased rate of weight gain, which
provides better Eeed conversion, which provides higher
yield of breast meat, and which provides for
uniformity of flock weight within the range of normal
size.
An even further primary objective of the present
invention is to provide direct-fed microbials suitable ;~
for poultry feed ration addition which contains
bacteria that are in microsphere form using a special
rotary technique using free fatty acid matrix.
Another objective of the present invention is to
provide a direct-fed microbial which has stability at
levels within the ran~e of from 3 months to 6 months
without any significant organism count reduction.
Another objective of the present invention is to
provide a process of rotary formation of spheres
containing!the dried bacteria which provides having
uniform size.
Another objective of the present invention is to
prov~dQ rotary diSC spheres of dried bacteria which
are free flowing, and easily processable with poultry
feed rations.
~093/1~162 2 1 3 ~ 7 9 0 PCT/US93/00867 ''
A still further objective is to provide a
microsphere of fatty acid material containing certain
bacteria, with spheres being useful as a direct~fed ;~
microbial for both poultry and swine. ~'
B~IEF DESCRIPTION OF TE~E DRAWINGS
Figures l, 2 and 3 show graphically the stability ~,
of the strains using stearic acid matrix.
Figure 4 is a graph showing breast yield
distribution for a feeding trial of the direct-fed
microbial composition of the present invention.
Figure 5 is a graph showing body weight ~'-
distribution for a feeding trial of the direct-fed
microbial composition of the present invention.
Figures 4 and 5 show a control, use of an
antibiotic and use of the direct-fed microbial of the
present invention.
. .
SUMMARY OF THE INVENTION ;
The invention is a method and composition of
growth promotion for poultry and swine which comprises ,
adding to the normal feed ration a small but growth
promoting effective amount of a direct-fed microbial
which contains dried, fatty acid microspheres of
Enterococcus faecium 301, DSM No~ DSM-Nr. 4789, and -
dried fatty acid microspheres of Enter,,o,coccus faecium
202, DSM No. DSM-Nr'. 4788, where preferably the fatty
microspheres are formed by rotary disc drying.
DETAILED DESCRIPTION OF THE INVENTION
It has been surprisingly discovered that the
growth promotion of poultry and swine can be
accomplished ~y adding to normal feed rations, a ,,
. .
WO93/19162 PCT/US93/008fi'
~131790 - 6 -
certain amount of fatty acid microspKeres of
Enterococcus faecium 301, DSM No. DSM-Nr. 4789, and a
certain amount of fatty acid microspheres of
Enterococcus faecium 202, DSM No.-~SM-Nr. 4788. A
fatty acid employed may be any one of the Cl2 to C24
free fatty acids, but is preferably stearic acid. The
organisms are preferably present in about equal
am~unts but may vary within the range from about 30%
to about 70~ of one of the organisms with the balance
being the other.
It is not known precisely why these two organisms
prov$de ~e desirable features of the present
invention, especially for poultry, namely increased
rate of weight gain, better feed conversion, incre~ased
yield of breast meat, and increased uniformity of
flock weight. The fact is that they do, provided that
both are used in combination so that they can somehow
interact with each other, and providing that they are
used within the range herein expressed. It is these
combinations of features which some how interact and
co-act to provide the desirable features of the
present invention which allow significantly improved
poultry carcass, meat quality and processing. Similar
results can be achieved for swine as indicated by the
examples~
The amount of direct-fed microbial added to the
feed ration can vary considerably but generally will
be within the range of from about 0.5 pounds to about
~2.0 pounds per ton of feed, generally from about 0.8 -
pounds to about l.2 pounds per ton of fead, and
typically at about l pound per ton of feed. The
organism count, that is the number of colony forming
units per gram present in the direct-fed microbial can
~"093/19162 21 317 9 ~ PCT/US93/OOg67
also vary within the range of from about 1 x 106
CFU/gm to about 2 x 109 CFU/gm, but is preferably at ~:
about 2 x Io8 CFU/gm.
When the direct-fed microbial as previously
described is free ch~ice fed in the animal feed
ration, the combination of two strains of organisms
herein mentioned, behave as a growth promoter. Growth
promoters now used include antibiotics such as
Stafac ~ and BMD. The advantages of sub-therapeutic
levels of antibiotics as growth promoting additives
can be achieved with naturally occurring organisms of
the present invention provided that direct-fed
microbial ~s made in accordance with the present
invention and added in accordance with the method
described herein. In fact, there have been some
trials that suggest that a combination of direct-fed
microbial and growth promotant together exceeds the ~`
advantages of either alone and thus they may be used
together if desired. However, in most instances, it
is preferred to use the direct-fed microbial alone
since one of the objectives of the present invention
is to avoid use of growth promotants altogether.
The method of processing of the organisms is not
critical as long as the organisms can be kept alive to
delivery to the animal, and placed in a form so that
it will combine with animal feed well and is of a
generally uniform size so that dosage may be
controlled.
A preferable means of achieving these
requirements is by providing the organisms in a
microsphere of a fatty-acid matrix. A microsphere
refers to a fatty acid matrix in which a plurality of `~
organisms is lncorporated. It is different from a
WO 93/19162 2 1 3 1~ 9 PCT/I ~93/008~7
microcapsule in which individual organisms are each
encapsulated. In a microsphere the fatty acid matrix
functions for the composite similar to the
relationship between a cookie dough matrix and
chocolate chips, with the chips representi~g the
groups of organisms. This process is described in the
parent application of the co-inventor Rutherford, et
al. By this process, the bacteria are combined with a
heated fatty acid. The temperature of the fatty acid
and time of exposure of the bacteria to the fatty acid
is controlled to keep the bacteria alive, yet allow
mixing with the fatty acid. The mixture is placed on
a rotating rotary disk, with the result being a
microsphere of bacteria with a fatty acid acting as
the matrix. Several important advantages are achieved
using this method. First, the bacteria are kept alive
through the processing; second, the process combined
with the rotary disk technique allows for a uniform
size of the microsphere for improved dosing. Third,
the nature of the matrix, a fatty acid, allows the
formation of the unique microspheres. T~e combination
of the factors provides for a highly stable direct-fed
microbial with maximum effectiveness.
In the process of the parent application it is
important to note microspheres are formed wherein each
sphere constitutes a plurality of bacteria in a free
fatty acid matrix rather than an, individual
microencapsulator of each bacteria in a coating or
film like layer of fatty acid. This provides
stability advantages, and more effective dosin,g with
the bacterial treatment.
The preferred matrix agent is a C1~ to C24 free
fatty acid. While mixtures of fatty acids may be
~VO 93/19162 2 1 3 1 7 9 0 PC~r/US93/00867
employed, it is preferred that a single pure free
fatty acid be employed. It is also preferred that the
free fatty acid be an unsaturated fatty acid, with
the most preferred being stearic acid.
Generally speaking, it is important that the
fatty acid have a melting point less than 75C,
preferably within the range of 40~C to 75C. It must, -~
of course, be solid at room temperature in order to be
an effective matrix. All free fatty acids falling
within the range of chemical description heretofore
given will meet these requirements.
In order to enhance the product stability, the
bacteria are typically freeze-dried bacteria as
placed in the product. Thus, they can be revived by
moisture addition.
In the microsphere, made in accordance with the
process discussed below, the microspheres generally
comprise from about 50% to over 90% by weight of the
fatty acid component with the balance being bacterial
culture. The preferred range is from about 60~ to
about 75% fatty acid. If too little fatty acid is
used, the matrix will be inade~uate for protection.
On the other hand, if too much is used, the matrix
will be too thick and results in inadequate release
in the gut. -
The process as used in this invention is a rotary
disc microsphere formation process. Generally
speaking in the rotary disc technology, a slurry of
~the bàcteria and fatty acid components are thoroughly
mixed with the mixture beiny added at a uniform rate
onto the center of a rotating stainless steel disc.
It is there flung o~twardly as a result of centrifugal
force and forms a microsphere. It is then collected
WO 93/19162 PCI~US93/008~7
213179
-- 10 --
in a cooling chamber maintained at ambient conditions
or slightly lower, sized and readied for packaging.
While rotary disc encapsulation per se is known,
it is not known to make microspheres contained in a
matrix without a surrounding shell, nor is it known to
use the microsphere process or encapsulation with
free~e dried bacteria. Generally speaking, for
descriptions of rotary disc encapsulation, see a paper
by Johnson, et al. of the Southwest Research Institute
of San Antonio, in th~ Journal of Gas Chromotography, -~
October, 1965, ~ages 345-347. In addition, a rotary
~-disc machine suit~ble for use in this invention is
described in detail in United States Letters Patent,
Spark~, 4,675,140, issued June 23, 1987 and entitled
"Method For Coating Particles For Liquid Droplets"
the disclosure of which is incorporated herein by
reference. However, it is the process described in
the parent application that is most preferred.
It is important to note that rotary microsphere
formation provides a distinctly different product than
either conventional tower spray drying or
microencapsulation. In conventional tower spray
drying there is a tendency for particles to cluster,
for the coating to be uneven, and thus for the
stability of the product to be significantly effected
perhaps from days to weeks. This process provides a
shell coating around an object, and bacteria have
proven to be too small, too hard to keep alive or
provide in a uniform size to be of practical
usefulness. With microsphere formation, particularly
with agents used in this invention is used, the
stability of the resulting bacteria, even when
sub~ected to some moisture and antibiotics, will be -
'"~
~,,.
`~'093/19162 2131 7 9 0 PCT/US93/OOX67
for from ~hree to six months wi~h the viability of the
bacteria maintained in evenly distributed particles.
When the free fatty acid microspheres of the
present invention are used within the ranges
hereinbefore expressed, the rotary disk, typically
employing a 4"-6" rotary disc, can be run at the rate
of from 2000 rpm to 4000 rpm, preferably about 2500
rpm to 3200 rpm with a feed rate of from 50 ~rams to
200 grams per minute. The preferred conditions pres-
ently known are use of stearic acid, use of two
hereinbefore descrlbed organisms, a four inch rotary ~.
disc, 3000 rpm and a feed rate of lOO grams per minute
with a bacteria/stearic acid slurry of 35% bacteria,
65% stearic acid. When this is done, a product having -
a particle size of from 75 microns to 300 microns will -:.
be achieved, with a preferred level of less than 250
microns.
The following examples are offered to further ~:
illustrate, but not li~nit, the process of the present :~
invention. Some of the examples are described in :.;
connection with Figures l, 2 and 3. Examples l
through 4 and Figures l, 2, and 3 relate to the ~
invent~on of my prior case. Example 5, and tables 2- ;
lO, relate to the process of this present invention
for a poultry direct-fed microbial. Example 6 relates . :
to turkeys specifically and example 7 relates to
swine.
Example l
Example l correlates with Figure l. It shows the ~:~
product stability of two different strains of
Enterococcus faecium with te~peratures of 4~C and
.
27C. AS illustrated in Figure l, it shows a
WO93/1~162 PCT/US93/008~
~311 9 12 -
stability of the encapsulated strains of Enterococcus
faecium, with the encapsulation being by the rotary
disc device using stearic acid with a level of 35~
culture weight. Conditions of microsphere formation ~.
were as previously described herein, namely a 35/65
bacteria s~earic acid slurry at a temperature of 60C,
using a four inch rotary disc, operating at 3000 rpm
and a feed rate of lO0 grams per minute. The spheres
were formed, placed in heat sealed vapor barrier
pouches and destructively sampled weekly for CFU de- :
termination. It can be seen that the product of the
invention maintained excellent organism colony forming
unit (CFU) counts out to storage times at long as 70
days.
Example 2 :
. ._
Example 2 is to be in~erpreted in connection with
Figure 2. The figure shows the stability of
individual microsphered strains when mixed in a . ~:~
typical feed ration in the presence of three poultry
antibiotics. The ration consisted of the ~ollowing:
54% fine cracked corn -
Z6% soybean meal
2% fish meal
l.5~ dicalcium phosphate
1% limestone
5.5~ soy oil `
.,
12~ moisture conten~
~Three an~ibiotics were added at the following
inclu~ion rates by weight: decoquinoate 6% (454 ppm), ~-
salinomycin (50 ppm) and monensin sodium (120 ppm).
Cult~re was added to the mixture at a level to
deliver approximately lxlO CFU/gm feed. Feed was
213179~
~093/19162 PCT/US93/00867
packaged in heat sealed bags and ~ncubated at room
temperature. Samples were taken weekly for CFU
determinati~n. The graph of Figure 2 illustrates the
excellent stability.
Example 3
Example 3 is to be interpreted in conjunction
with Figur~ 3. It shows the stability of the
Enterococcus faecium microspheres in feed in the :~
presence of different antibiotics. The ration
consisted of 60~ fine cracked corn, 38~ soybean meal
and 2~ limestone with a moisture content of a~out 14
~- Culture was added to a level of approximately 106
CFU/gm feed and mixed. Ten pound aliqu~ts were stored
in sealed bags at 20 C and sampled weekly for 16
weeks. The ant~biotics were included in the ration
at the following levels: ::
Bacitracin methylene disalicylate ....... 50 gm/ton
Carbadox ................................ 50 gm/ton
Chlortetracycli.ne ..................... 200 gm/ton
Lasalocid ........................... ~... 30 gm/ton
Lincomycin ..........~.................. 100 gm/ton
Neomycin .............................. . 140 gm/ton
Oxytetracycline ....................... . 150 gm/ton
Sulfamethazine ........................ . 100 gm/ton
Tylosin ............................... . 100 gm/ton
Virginiamycin ......................... .. 20 gm/ton
ASP250 ................................ . 100 gmtton
Furadox ............................... .. 10 gm/ton
Table 1 is a list of the minimum times for a 1
log loss in colony forming units (CFU).
W093/19162 PCT~US93~00g~7
~3~90 - 14 - ~
Table l
Time in weeks for loss of l l-og cFu counts
at 200C in 14~ moisture mash feed. ;;
AntibioticTime of Storage fdays3
Control 103
Bacitracin ~8
Carbadox 54
Chlortetracycline 60 ~`
Lasalocid 57
Lincomycin' 75
~_- Neomycin 53 ;
Oxytetracycline 59
Sulfamethazine 62
Tylosin 52
Vir~iniamycin 112
ASP250 67
Furadox 53
Example 4
In Example 4 the stability of product after
pelletizing for use of a chicken feed product was
determined. The microsphere formation conditions were
as earlier described. The conditions used in this
study were the following:
Crude Protein, not less than .. 18.0%
Crude Fat, not less than ...... ~......... ....... 5.0%
Crude Fiber, not more than .............. ...... 6.0% ~;
The pellets with and without the antibiotic (CT~
50 gm/ton) were made with the following ingredients
and conditions.
Corn, SBM, whey, soy oil, dicalcium phosphate,
limestone, trace mineral premix, vitamin premix, -~
'~
..:..
~'093/19162 213 t 7 9 ~ PCT/US93/00867
- 15 -
selenium, copper sulfate. Culture was added at
approximately 5x105 CFU/gm feed.
Conditioning temperature was 70C and the pellets
out of the dye were 78C.
Pellets were stored in unsealed bags and sampled
weekly for CFU determination. ;~
In each instance the pelletized product was not `
adversely affected in stability by the conditions of
pelletizing. In particular, the pelletized product
showed stability equal to the unpelletized product.
~r Example 5
Four thousand five hundred sixty, day-old
Peterson x Arbor Acres broiler chicks were randomly
assigned to floor pens (Tabla 2) with reconditioned
litter and fed for 45 days. All birds dying during
the f~rst 5 days were replaced with a same-sex bird
from the same shipment and same treatment. The
composition of the basal starter, grower, and
withdrawal rations is shown in Table 3. Starter,
grower, and withdrawal rations were formulated to
contain 1425, 1450, and 1475 kcal ME/lb, respectively,
with 90 g/ton monesin. Starter rations were fed from
l to 21 days of aye, grower from 21 to 42 days of age,
and withdrawal from 42 to 49 days of age. The
treatments were negative control, mash ~Control, M);
a selected, encapsulated direct-fed microbial cultures
containing Enterococcus faecium 301, DSM No. DSM-Nr.
4789 and~Fnterococcus faec1um 202, DSM No. DSM-Nr.
4788 each rotary disc fatty acid encapsulated as
described in Example l and each present as 50% of the
direct-fed microbial applied at l x 105 CFU/g of feed,
mash (direct-fed microbial, M); negative control, ;
WO 93/lgl62 PCr/US93/0086?.
~3i79 16 -
pelleted (Control, P), direct-fed microbial applied at
1 x 106 CFU/g mash, pelleted (direct-fed microbial,
P), and a positive control applied at lQ g~ton
virginiamycin, pelleted (Stafac ~ 10). The starter
ration was crumbled for the treatments that were
pelleted. Twelve replicated pens of 35 males and 35
females were used with each experimental ration. ~
Body weights, feed consumption, and mortality ;
after the first 5 ~ays were recorded by pen. Feed
conversion, adjusted feed conversion, and body-weight
ad~usted feed conversion were calculated for each pen.
~- All data were subjected to analysis variance and
differences were determined using Fisher LSD.
Prior to the study, direct-fed microbial culture
concentrate was extended with calcium carbonate. The
theoretical counts for direct-fed microb~al, M and
direct-fed microbial, P were 1 x lO8 and 2 x lO9 CFU/g
of product, respectively. An 11 9 sample of each
product was assayed in duplicate to determine actual
prodùct counts. Each sample was plated using the
standard plating technique for encapsulated lactic
acid bacteria.
A mixer test was conducted for each production
phase. The test was designed to ensure that the
direct-fed microbial was uniformly distributed at
appropriate levels in the feed and that it survived
pelleting. Each batch was sampled at the time of
bagging with 4 equally spaced samples for the mash
~treat~ents and 10 equally spaced samples for the
pelleted treatments (i.e. bags 1, 3, 5,... , 35, 37, ~
and 39). ~;
Alternate floor pens within a treatment had non-
contaml nated feed sampled during weeks 1 and 4; with
'`'093/19162 2 1 ~ 1 7 9 o PCT/US~3/00867
- 17 -
the remaining pens sampled on weeks 2 and 6 during the
feeding study.
An equal number of birds from each sex was
sacrificed for the determination of individual breast,
body and small intestinal weights, and small
intestinal length. Breast yield and intest~nal weight
and length ratios were calculated for each bird.
All data were sub~ected to a split-plot analysis
of variance and differences were determined using
contrast and est~mate statements for the desired
effects.
~~ Sixty birds per treatment were transported to a
university for a sensory taste panel evaluationO
Direct-fed microbial, regardless of processing,
improved (P<.05) feed conversion over the respective
Control while increasing (P<.05) weight gain over the
Control only in the mash feed (Table 4). The direct-
fed microbial, P improved (P>.05) feed conversion over
Stafac ~ 10 which was similar (P>.05) to Control, P.
The product was at its desired level and strain
composition (Table 5).
Direct-fed microbial was uniformly distribu~ed
within the feed. Direct-fed microbial, M was at its
desired level while direct-fed microbial, P was 1 to
1-1/2 log higher than desired for the starter and
grower rations (Table 6). The high counts for direct-
fed microbial, P were a result of overengineering of
the product to ensure sufficient recovery of the
organismslafter pelleting.
The floor pen samples for the direct-fed
microbial, P corresponded closely with the counts from
the mixer tests (Table 7)~ However, d-irect-fed
microbial, M dropped 2 logs in weeks 4 and 6 in the
grower and withdrawal mixes.
WO93/19162 PCTJUS93/00867 `~
21~179
- 18 -
Direct-fed microbial, M increased (P<.05) both
breast weight and yield over the Control, M (Table 8)
while direct-fed microbial, P showed an improvement
(P>.05) over Control, P. The improvement in the mash
feed agrees with the results found in an earlier
trial. The direct-fed microbial, P did not show a
similar magnitude in improvement in breast yield to
that observed in direct-fed microbial, M. This
failure may be due to improved energy utilization by
pelleting resulting in less room for improvement.
Pelleting increased the average bird weight by 96
g over mash. Direct-fed microbial increased the
uniformity of bird weights (Figure 5) with the
greatest improvement is mash feed.
Pelleting increased the average breast weight by
15 g over mash. Direct-fed microbial increased the ;~
average breast weight and uniformity (Figure 4) over
the Control with the greatest improvement found in
mash. Stafac ~ 10 showed the greatest improvement in
uniformity for the pelleted feeds.
Pelleting increased breast yeild by .53
percentage unlts over mash. Direct--fed microbial, M
showed a .84 percentage unit increase over Control, M
which was similar in magnitude to the pelleting
respoonse.
The direct-fed microbial treatments produced a
shorter (P~.05) small intestinal length than either of
the Controls and Stafac ~ when expressed as actual
length, a ratio of either body weight, or breast ~ ;
weight (Table 9). Direct-fed microbial, M had a
lighter (P>.05) small intestinal weight than Control,
M when expressed as either actual weight or percentage
of either body or breast weights. The reduction in
~/093/19162 2 1 3 ~ 7 9 o PCT/US93/00867
- 19 -
intestinal weight and length for direct-fed microbial
treatments suggests less energy required for
maintenance and more energy available for growth as ::
indicated by improved feed conversion and breast yield
(Table 7-8).
The direc~-fed microbial, P treated birds
produced no off-flavor when compared to Stafac ~ 10
(Table 10). In the second trial, direct-fed
microbial, P was perceived to have enhanced the flavor
of th~ thigh/leg when compared to Control, P.
However, this enhancement of flavor was not observed
~-in the first trial.
WO 93/19162 PCI/US93/0086
~,1317gO 20
TABLE 2
PEN AsS~ r
.
~.~
; .
TreatmentsPen numbers
Control, P 2,6,15,17,'2,26,10~,109,113,11?,122,126
Dircct-red microbial.l' 4,8,12,16,21,2~,105,106,112,118,125,130
Stafac~ 10 5,7,11,18,23,'7,101,107,111,116,123,129
Control, M 3,9,13,2~,24,30,102,10~,114,119,121,127 ~:
~irect-fc~ microbial,~l 1,10,14,19,25,~9,103,110,115,120,124,128 ~:~
:
Pen size 4.2' x 15.5', one tube feoder, one hanginy waterer, pine
shavings on dirt, power and evaporative cooling system and well
insulated, forced h~t-air heat, curtain sidewall building.
TABLE 3 :~
COMPOSITION OF BA5~L RATIONS
.
Production Phases
ln~redientsStarter Grower Wi~drawal
: .
%
Ground corn65.37 67.89 74.29
Soybean meal25.58 23.53 17.83
Meat and bone meal 3.00 3.00 3.00
Fat 3.36 3.32 2 . 59
Deflourinated phosphate.5 .79 .73
Ca1cium carbonate .61 .62 .63
Salt .35 .31 .32
Trace mineral.05 .05 .05
Methionine. 39 . 2B . 33
L~sine .19 .06 .18
Vitamun premix .05 .05 05
.
.,
: .
~vo 93~19162 2 ~ 3 ~ 7 9 o P ~ /US93/00867
- 21 -
IAsLl,4
FI~OE~ N PI~ODUCTION at~T~
:
Pellet Mash
Inv r stafac~ Inv
Control P lO Control11
_ - :
Weight, lb. 4.79~ 4.8l~ 4.79~ 4.54b 4.6~
Feed conv. l.871l.827A l.855 l.9l7el.056
~dj. feed conv.1 1.832 1.7~9 1.~07 1.8~7' 1.~12
Weight, ad~. ~ b
feed conv.2 l.80lb 1.755 1.775l.897C 1.798
Mortality, ~ ' 4.4~ 4.64 5.95 3.33 5.60
~dj. feed conversion ~ ~otal feed/(live + dead weight).
2 we~gllt ad~. feed conver610n - ~dj. feed conversion-((weigl1t-4.60)/6).
P<.05. ::
* lnv ~ lnvention ~:
T~LE 5
PRODUCT QC ~D QA
.
Treatments QC~count QA count Strain ratio
~~ - cfu/g of product -- s~202:5~301
~irecL-fedmicrobial.l' 5.75 x lOa 1.01 x 10~ 50:50
~irect-fed micr~ia~ 9.S4 x 107 - 9.6~ x 107 57 43 :~
.. .
1. Quality control
2. Quality assurance.
WO 93/19162 PCI/US93/00867-
L3~g `~
-- 2 2 -- -
~;.~,,
l'ABl.E 6
FEEDt~ILL MIXER TEST r~NC? RECt~ nY . '
':
Production Phases
and Treat~?ents Masi- Pellet Recover~
cfu/g of feed - - ~ mash -
Starter ~`
Control, P N~,2 1 . 06 x 103 - : `
Dlrect-fec? microbial,l> 2.02 x 1061.67 x 106 9B.69
Stafac~? 10 NA 6.~6 x 103 - `.
Control, M 2.51 x 103 ~ `
Vire?~t-rednlicroL~?i~l 1.34 x 105
Grow?er
Control, P N~ 4.86 x 102
Dlrect-fed? microbial,r 3.es x 1061.09 x 106 91.~?2
Stafac~? 10 5.25 x l01 fi.42 x 103
Control, M 1.50 x 10
Dir~ct ~ed? microb?ial,M 1.48 x 10
Wlthdrawal
Control, P 6.50 x 102 1~11 x 103 - ~,
Direc~fedlmicrol)~?l,P 7.04 x 1044.91 x 105 117.40
Stafac 10 6.B0 x 10 1.7S x 10 -
Control, M B.92 x 102 -~:
~irect fetl? micro?~?iai ~ . 3 .? X 10
Mean
Conteol, P 8.50 x 102 B.28 x lC2 -
Direct fe~? nùcrob?ial ~ I a . 21 x 10 9.64 x 10 llE?.09
Stafac~? 10 2.15 x 10~ 9.05 x 10~ -
Control, M E.72 x 10
DireCt-f~? microbial,~l 1.3e? X 105 .,.;;
,.:
Recovery calculated on log10 transformed data.
N~ means not available.
! ~
'``' ~
~'
~`'O 93/19162 2 131 7 9 O PC~r/U$93/00867
1-~13LE 7
~LOOR PEN
Weeks
TreatmRnts 1 2 4 ~ Mean
-- cfu/g o~ ee~
Cont~ol ~.,8 X 102 3.83 X 10~ 8.60 x 10~ 2.21 x 10~ 4.08 x 102
Direct-r(~lmicrobiçl,P 9.23 x 105 9.~7 x 1058.77 x 105 8.48 x 105 a.96 x 105
Stafac 10 8.73 x 102 1~9 x 102 6.46 x 10~ 8.63 x 102 8.89 ~ 102
Contsol, M 3.46 x 102 1.~6 x 102 2.79 x 103 2.00 x 102 5.08 x 102
L~irect-f~lmicro~ial,M 1-43 x ~05 1.'5 x 1051.75 x 10~ 1.00 x 10~ 2.32 x 10
. .
TA~LE 8
BRE~ST YI~LD F~ALUATION
Pellet Mash
lnv~Ç Sta~ac~ --~~--
Control P 10 Control
Body weight, g 2240.72230.1 21g5.9 2143.82149.9
Breast weiollt, g 234.4' 239.6' 232.~' 213.3b 22g.6'
~reast yield, S of
body weight 10.51~ 10.68' 10.58' 9.93b 10.67'
. .
ab P<.0;
~Ç Inv ~ Invention
W 0 93/19162 PC~F/US93/00867
~ ~3~ 9 ~
- 24 -
T~L~ 9
It~TES'rINAL WE:IGIIT ~ND L~,n~
:
Pe11et Mash
Inv ;~ Stafac~ -~~~-~lnv ;~
Contro1 P 10 Control M
Pody weigltt, 9 2240.7 2230.1 2195.9 2143.~ 2149.9
~reast we$ght, 9234.4~ 239.6- 23Z.o~ 213. 3b 229.6
S~ weigllt, g 92.6 93.3 93.~ 91.4 81.4
SI length, in 76.3 75.3 76.6 76.1 75.3
SI, g/in 1.21 1.23 1.22 1.20 1.16
SI weight, g/100 g
body weigllt 4.17 4.1~ 4.27 4.29 4.0
SI length, in/100 9
~-~ body weigllt 3.47 3.40 3.53 3.61 3.53
SI ~eight, 9~OO g
breast weight 40.1g 39.70 40.97 43.96 3B.69
SI length, in~100 9
breast weight 33.41- 32.27^ 33.72~ 36.89b 33.41-
:
ab P<.05
* 1nv ~ Invention
~I a Small Intestille ;-
T~LE lO
TASTE PN ~L F~ALUA~IOt~
~ - ~.
Group Number of correct ioentifications
Tissue Comparison TriaI 1 ~ ~rial 2combined
.;
Thigh/leg Stafac~ 10 vs. Control, P 5 3 9
Stafac- 10 vs. XIt~o~, P 3 4 7
~irect-fed microbial,~' ~s. Control. r 2 8~ 10
sreaSt 5tafat~ 10 vs. Control, P 2 6 B
Stafac~ 10 vs. XINOC, P 1 3 4
~irect-fed microbial,~' V5. Control, P 5
~he evaluators were able to detect the odd sample a statistically -~
sionificant ~ P< . 05 ) nwnber of times .
~he nwnber of correct identifications of the odd samDle required for
si~niflcance at the 5% level was 7 for n-10 and 11 for n-200
`~
;,
. .
3~ } ~ p--~q~ ;~ ' ~ ` i ~
''O 93/19t62 2 ~ 3 1 7 9 0 PCT/US93/008~7
I
- 25 -
A broiler trial was conducted to determine the
efficacy of direct-fed microbial in mash and pelleted ~
feeds. Direct-fed microbial, regardless of ;
processing, improved (P<.05) feed conversion over the
respective Control while increasiny (P<.05) weight
gain over the Control only in the mash feed. Direct-
fed microbial, P improved (P>.05) feed conversion over
Stafac ~ 10 which wa~ similar (P>.05) to Control, P.
Direct-fed microbial, M increased (Pc.05) both breast
weight and yield over Control, M while direct-fed
microbial, P showed an improvement (P>.05) over
Control, P. Direct-fed microbial, P treated birds
produced no off-flavors when compared to Stafac ~ 10
treated.
Example 6
One hundred forty four, commingled feedier pigs
(average initial weight 41.5 lb) were randomly
assigned to slated-floor pens by weight and sex (Table
ll) and fed fnr ll9 days. The composition of the
basal grower and finisher rations is shown in Table
12. Grower rations were fed until individuial pens
averaged 120 lb followed by the finisher until
slaughter. All dSets contained Mecadox ~ (50 g/t) up
through 75 lb bodyweight followed by 100 g/~
chlortetracycline until 120 lb liveweight. The
treatments were negative control (Control) and a
selected, microsphered direct-fed microbial cultures
applied at 1 x 10 Gfujg of feed. All rations were
fed in mash form. Six replicated pens of 12 feeder ~-
pigs were used with each experimental ration.
Upon arrival at the research facility, the
commingl~d feeder pigs wexe given Ivomec ~ to control
internal and external parasites. Safeguard ~ was
given after four weeks to control whipworms.
W093/19162 PCT/US93/OO~fi?
2 1L3~rl 9~
- 26 -
Body weights, feed consumption, and mortality -~
were recorded by pen. Feed conversion was calculated
for each pen.
Prior to the study, the microsphere culture
concentrate was extended with calcium carbonate. The
theoretical count was 2 x 10 cfu~g of product. An 11
g sample of product was assayed in duplica~e to
determine actual product counts. The sample w2s
plated usin~ standard plating techn~que fox
microsphered lactic ac~d bacteria.
An additional 1 ~ sample of product was assay~d
in dupllcate to verify the product count and strain
composition.
Samples were taken for each treatment weekly and
tested for microsphered lactic acid bacteria.
The product was confirmed as at its desired
organism level (Table 14). `~
Pen sample recoveries varied ~rom 1 x 101 to 1.6
x 105 cfu/g of feed (Table 15). The two extreme
samples are attr~buted to sampling/plating error. The
remainder of the samples averaged around the target
level of 1 x 104 cfu/g of feed.
Microsphered product improved (P>.05) weight gain
and feed conversion over the Control after 28 days
(Table 13). The pigs were hit with a TGE outbreak the
first week of the trial. This outbreak along with
time required for pig's digestive tract to adjust to
the product may be why a 28 day lag was observed prior
to an observed response. It can then be seen that~the
microsphere's of direct-fed mi~robial of this
invention function effectively for swine as well as
chickens and turkeys. ~-~
'~1093/19162 213 1 79 0 PCT/US93/00867
- 27 -
From the above examples it can be seen that the
invention accomplishes each of its sta~ed objectives.
TABLE ll
PEN ASSIGNMENTS
Experiment: 670-9102
. . .
Treiatmentis Pen numbers
Control 3, 4, 6, 9, ll, 12
Direct-fed ~icrobial l, 2, 5, 7, 8, l0
* Pen size 4.6i x 16.0', one four-hole Smidley
feeder, one nipple driker, sprinklers were used
for heat control, partially-slated floor, and .-:
modified, open-front building. -
Table 12 .~.
COMPOSITION OF BASAL RATIONS
Experiment: 670-9102 ~
Ingredients Production phases ;
Grower Finisher
Ground corn 76.30 82.20
Soybean meal 21.25 15~50 ~
Dicalcium phosphaste l.05 ~90 --
Calcium carbonate .85 .85 .:
SaIt .30 30 .
Vitamin/mineral premix.25 o25 i
. .
: :`
~,,.
WO 93/19162 P~,~/US93tO08~7
~,~3~9~ ` :
;~ 8
Table ~3
FLOOR PEN PRODUCTION DATA
Experiment: 6 7 0 - 9 l 02
: .~
Direct-Fed
Control Microbial
Day 14
Weight gain, lb. 9.6 9.2 ~.
Feed conversion 2.439 2.483 ::
. Mortality, ~ 1.41 1.39
Day 28 ~.
Weight gain, lb. 26.3 ~7.9
Feed conversion 2.405 2.212
Mortality, ~ 2.82 1.39
Day 42 . -~
Weight gain, lb. 45.8 46.8
Feed conversion 2.497 2.428
Mortality, ~ 2.82 2.78
Day 56
Weight gain, lb. 69.1 71.5
Feed conversion 2.507 2.457
Mortality, % 4.23 2.78
Day 70
Weight gain, lb. 89.4 91.2
Feed conversion 2.735 2.674 ~
Mortality, % 5.63 2.78 :
Day 84
We~ght gain, lb. 111.0 112.0 ~:
Feed conversion 2.504 2.882 ~.
Mortality, %; ~5.63 ~2.78
Day 98
Weight gain, lb. 12~.9 134.7
Feed aonversion 3.071 2.988 ~:
Mortallty, % 5.63 2.78 ~
`~093/19162 2 1 3 ~ 7 9 o PCT/US93/00867
- 29 - :
Day 112
Weight gain, lb. 152.2 154.7
Feed conversion 3.164 3.134 :~
Mortality, ~ 5.63 2.7B
Day 119
Weight.gain, lb. 162.4 165.6
Feed conversion 3 . 217 3 .177 :
Mortality, ~ 5.63 4.17
Table 14
PRODUCT CQ AND CA
Experiment: 670-9102
,. '~
: .
Product QC count QA count Strain Ratio
cfu/g of product SF202:SF301
Direct-Fed Microbial 4.3 x 107
,,;
WO 93/19162 PCl'/US93/008
rl9
-- 30 --
Table 15
FLOOR PEN QA
Experiment: 670 - 9 1 ():2 ~ :~
- -- :
Direct-Fed
Date Control Microbial
5/3/91 1.7 x 1034 7.4 x 103
5/8/91 l.~ x 10 1.~ x lOl
5,t22~91 0 1.0 x 103
6/5/99~ 1 o x l1 9 6 x 1o3
6/26/91 6.7 x 12 5.6 x 103
7/3/91 4.9 x 10 3.2 x 104
7/10/91 5.2 x 101 3.0 x 103
7/17/91 1.~ x 102 4.5 x 104
7/24/91 0 1 2 x 104
8/l4/gl 0 1 1 x lo4
9/4/91 0 2.9 x 3
9/18/91 0 8.9 x 103
9/25/91 0 5.5 x lO
Mean 9.5 x 10 8.4 x 103
