CA2131790A1

CA2131790A1 - Fatty acid microspheres containing enterococcus for use to enhance growth and improve carcass quality

Info

Publication number: CA2131790A1
Application number: CA002131790A
Authority: CA
Inventors: William M. Rutherford; Jack E. Allen; Scott M. Dennis; Mark A. Hinds; Gregory R. Dana
Original assignee: Individual
Current assignee: Pioneer Hi Bred International Inc
Priority date: 1992-03-17
Filing date: 1993-02-03
Publication date: 1993-09-30
Also published as: HUT67965A; SK111694A3; RU94043791A; MX9301017A; RO112896B1; JP2849877B2; EP0631616A1; WO1993019162A1; BR9306121A; CZ225394A3; RU2109052C1; JPH07505056A; EP0631616A4; SI9300128A; HU9402673D0; BG99113A

Abstract

Dried, rotary disc fatty acid microspheres of Enterococcus faecium), strains 301 and 202 are mixed and used as a feed additive for animals for growth enhancement and carcass quality improvement.

Description

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 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 ~ .
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 .

2 P~T/US93/008~
2131~790 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 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 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 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 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 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 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.

~,1317gO 20 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 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.

L3~g `~

~;.~,, 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 ~

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

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~

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 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 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

Claims

What is claimed is:

1.
A method of growth promotion of animals comprising adding to an animal feed ration a growth-promoting effective amount of a direct-fed microbial consisting essentially of viable, stable, dried fatty acid microspheres of Enterococcus faecium 301, ATCC No. 55059, and viable, stable, dried fatty acid microspheres of Enterococcus faecium 202, ATCC No.
53519.

2.
The method of claim 1 wherein the fatty acid microspheres are formed using a rotary disc.

3.
The method of claim 2 wherein the direct feed microbial composition is from about 30% to about 70%
of one of said fatty acid microspheres with balance being the other.

4.
The method of claim 3 wherein the fatty acid is a C12 to C24 free fatty acid.

5.
The method of claim 4 wherein the fatty acid is stearic acid.

6.
The method of claim 1 wherein the amount of direct feed microbial composition added to the feed ration is from about 0.5 lbs. to about 2.0 lbs./ton of feed.

7.
The method of claim 6 wherein the amount of direct feed microbial composition is from about 0.8 lbs. to about 1.2 lbs./ton of feed.

8.
The method of claim 6 wherein an organism count of the direct feed microbial composition is from about 1 x 105 CFU/gm to about 2 x 108 CFU/gm.

9.
The method of claim 8 wherein the organism count of the direct feed microbial composition is about 1 x 105 CFU/gm.

10.
The process of claim 1 wherein the animal is a chicken.

11.
The process of claim 1 wherein the animal is swine.

12.
A direct feed microbial composition for growth enhancement of animals consisting essentially of viable, stable, dried fatty acid microspheres of Enter coccus faecium 301 ATCC No. 55059, and viable, stable, dried fatty acid microspheres of Enterococcus faecium 202 ATCC No. 53519.

13.
The direct feed microbial composition of claim 12 wherein the fatty acid is a C12 to C24 free fatty acid.

14.
The direct feed microbial composition of claim 13 wherein the free fatty acid is stearic acid.

15.
The direct feed microbial composition of claim 12 wherein the animal is a chicken.
16.
The direct feed microbial composition of claim 12 wherein the animal is a swine.

17.
A method of claim 1 wherein said Enterococcus faecium 301 and said Enterococcus faecium 202 are present in about equal amounts.
18.
A direct feed microbial composition of claim 12 which has from about 20% to about 30% of said Enterococcus faecium 301 or said Enterococcus faecium 202 with the balance being the other of said organisms.
19.
The direct feed microbial composition of claim

16 wherein said Enterococcus faecium 301 and said Enterococcus faecium 202 microspheres are present in about equal amounts.