CA2116525A1 - Fatty acid microencapsulated enterococcus for use with poultry - Google Patents

Abstract

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

Description

~ W093/0620~ PCT/~IS92/07~X') 2116~25 -FATTY ACID MICROENCAPSULATED ENTEROCOCCUS
FOR USE WITH POULTRY
. , BACKGROUND OF T~E INVENTION
Growth enhancers in the form of antibiotics have been used extensively for poultry, namely chickens and turkey. Growth enhancers su~h as Stafac ~ and ~.
BMD ~ ~bacitracin methylene disalicylate) are known 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 objections 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 use~ in order to improve feed conversion, improve carcas:s composition, and enhance growth.
It is known that certain bacteria are potentially beneficial when added t5 animal feeds.
These ~acteria are beneflcial in that they supply a natural intestinal micro-flora. Some companies offer for sale probiotics which contain desirable ba~teria. Probiotics, however, do have some dif-iculty in maintaining a s~able product. Typically, the probiotic is used at a fairly low level, added to feed at perhaps a 0.1% level. However, unused probiotic containing feed or feed additive product is often stored by the farmers for long periods of time. This storage many times is under conditions where there is some moisture and high temperature.
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In many instances there is just enough moisture that the bacteria are activated or start to grow, but yet there is an insufficient amount of moisture to . .
sustain them. As a result they die. Thus, the acti~ity of the probiotic is stopped. In other instahces, ~he addition of antibiotics to the probiotic containing feed or feed additive adversely interacts wi~h the bacteria, particularly if there are small amounts of moisture present and thus again bacteria are killed. Thus, there is a significant problem of long term storage stability for probiotics.
In another environment, where the probiotic is added to, for example chicken eed, it is common to pelletize the material with the probiotic 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 i~activating bac~erla before they really reach the intestine. Thus, ~here is a continuing need for pr~bio~ics 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 intesti~e.
Certain features of poul~ry are especially de~irable ~o 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 course, desirable for the attendant economics ~hat accompany . W~93/06208 PCT/~IS92/075~'3 2116S2~

these desirable results. The composition of carcass is impor~ant because the most desirable area for tissue deposit is the breast in order to yield a hi~h amount of choice meat. Thus, weight gain is not only important, but where the weight is gained on the carcass ~s also important. Vniformity of flock weight ls important because if more birds are normal in size, less hand labor is required and processors j can more extensively rely on mach~ne processing. On the other hand, if the birds vary considerably from very small birds to very large birds, even though the ; overall flock weight may be the same, the smaller birds and the larger birds require a great deal more 31 hand labor and ~ecause of their lack of uniformity insize, ¢annot be processed easily by machine. Thus, ~ uniformity of flock weight wi~h a high percentage `~ distribution within the normal size range so that t chickens can process by standardized machinery is a .3 desirable feature.
`: : It is a~primary objectlve of the present l invention to provide a poultry probiotic which '3 ' contains no antiblotics and contains only fatty acid i ~ - microencapsulated:naturally occurring organisms.
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~3 It is another primary objective of the present invention to provide a probiotic which contains two ~ organisms, nameIy Enterococcus faecium 301, DSM No.
3 DSM-Nr. 4789, and Enterococcus faecium 202, DSM No.
~, I DSM-Nr. 4788. DSM is a Bacterial Culture collectio~
~ in Germany. DMS stands for Deutsche Sammlung von ,: Mikroorganismen located in Braunschweig, West Germany. These organisms will be deposited at the ATCC, with ~11 restrictions lifted upon notice of , /
.~: allowable claims.
L~ It is a further objective of the presen~
-~ invention to provide a probiotic which, for poultry, ..:
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provides increased rate of weigh~ gain, which provides better feed conversion, which provides higher yield of breast meat, and which provides for~
uniformity of flock weight within the range of normal slze.
An even further primary objective of the prese~t invention is to provide probiotics suitable for poultr~ feed ration addition which contains bacterla that are in microsphere form using a special rotary technique using free fatty acid matrix.
Another objective of ~he present invention is to provide a probiotic which has stability at levels within the range of from 3 months to 6 months without any significant organism count reduction.
~ nother objective of the presen~ invention is to provide a process of rotary formation of spheres of the dried bacteria which provides having uniform size.
Another ob~ective of the present invention is to provide rotary disc spheres of dried bacteria which are free flowing, and easily processable with poultry feed rations. -BRIEF DESCR~PTION OF THE DRAWINGS
Figures 1, 2 and 3 show graphically thestability of the strains using stearic acid ma~rix.
Figure 4 i5 a graph showing breast yield distribution for a feeding trial of the probiotic composition of the present invention.
Figure 5 is a graph showing body weight distri~ution for a feeding trial o~ the probiotic composition of the pr~sent invention.
Figures 4 and 5 show a control, use of an antibiotic and use of the pro~iotic of the present lnvention.

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W~93t0620~ P~T/~'S92/075X') 211~5~

~UMMARY OF THE I~VENTION
The invention is a method and composition of growth promotion for poultry which comprises adding to the normal poultry feed ration a small but growth promoting effective amount of a probiotic which contains dried, atty acid microspheres of Enterococcu$ aecium 301, DSM No. DSM-Nr. 4789, and dried fat~y acid microepheres of Enterococcus faecium 202~ DSM No. DSM-Nr. 4788, where preferably the fatty microspheres are formed by rotary disc drying.

DÉTAILED DESCRIPTION OF THE INVENTION
It has been surprisingly disco~ered that the growth promotion of poultry can be accomplished by adding to normal poultr~ feed rations, a certain amount of fatty acid microspheres of F.n~erococcus faecium 301, ~SM No. DSM-Nr. 478~, and a certain amount ~f fatty acid microspheres of ~nterococcus i fae~ium 202, DSM No. DSM-Nr. 4788. A fatty acid t employed may be ~ny one of the C12 to C24 free fatty acids, but is preferably stearic acid. The organisms are preferably present in about equal amounts but may vary within the range from about 30% to about 70~ of one of the organisms with the balance being the ' o~her.
'`3! ' It is not known precisely why these two -'~ organisms provide the desirable features of the ~ present invention, namely increased rate of weight :. gain, better feed conversion, increased yield of breast ~eat, and increased uniformity of flock weight. The fact is that they do, provided that both , are used in combination so that ~hey can somehow '~J in~eract with each other, and providing that they are -S used within the range herein expressed. It is these , combinations of features which some how interact and ,.
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co-act to provide the desirable features of the present invention whlch allow significantly improved poultry carcass, meat quality and processing.
The amoun~ of probiotic 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 1.2 pounds per ton of feedj and typically at ~bout 1 pound per ton of feed. The organism count, that is the number of colony forming units per gram present in the probiotic can also vary within the range of from about 1 x 10 CFU/gm to about 2 x 1~9 CFU/gm, but is preferably at about 2 x CFU/gm.
. When ~he probiotic as previously described is free choice fed in poultry feed ration, the " .
i combination of two strains of organisms herein mentio~ed, behave as a growth promoter. Growth promoters now used include antibotics such as i Stafac ~ and BMD. The advantages of sub-therapeutic ~: levels of antibioti~s as growth promo~ing additi~PS
. can be achieved with na~urally occurring organisms of ~t~ the present invention provided that probiotic is ~ade `. in acco~dance~with ~he present invention and added in i~ accordance with the me~hod described hereinO In fact, thare have been some trials that suggest that a " combination of probiotic 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 .; probiotic alone since one of the objectives of the `~ present in~ention is to avoid use of growth Z promotants altogether.
:.~ The method of processing of the organisms is not - critical as long as the organisms can be kept alive . q -.
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. W093/06208 PCT/~S92/07~
2116~5 -to delivery to the animal, and placed in a foxm so that it will combine with animal feed well and is of a generally uniorm size so that dosage may be controlled.
A preferable means of achieving these requirements is by providing the organisms in a microsphere of a fat~y-acid ma~rix. This process if described in the parent application of the co-lnventor Rutherford, Pt al. By this process, the bacteria are combined with a heated atty acid. The temperature of the fatty acid and time of exposure of the ~acteria 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 atty acid acting as the matrix. Several important advan~ages are achieved using this method.
First, the bacteria are kept alive through the processing; second, the process combined with the rotary disk technlque~allows for a uniform size of the microsphere for improved dosing. Third, the nature of the matrix, a ~atty acid, allows ~he fcrmatîon of the unique microspheres. The combination of the f ctors provides for a highly stable probiotic with maximum effectiveness~
In the process of the parent application it is important to note mic~ospheres are formed wherein each sphere consti;tutes 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 dosing with th bacterial treatment.
The preferred encapsulating agent is a C12 to C24 free fatty acid. While mixtures of fatty acids .' W093/06208 PCT/USg2/075X'3 2:~C~2~ `

may be 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 ~peaking, it is important that the fatty acid have a melting point less than 75~C, preferably within the range of 40C 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 chemica1 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 revi~ed by moisture addition.
In the microsphere, made in accordance with the proce~s discussed below, the microspheres generally compr~se from about 50% to over 90% by weight of ~he 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 wlll be inadequate 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.
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The process as used in this invention is a rvtary disc microsphere forma~ion process. Generally I speaking in the rotary disc technology, a slurry of tre bacteria and fatty acid components are thoroughly mixed with the mixture being added at a uniform rate i onto the center of a rotating stainless steel disc.
It is there flung outwardly as a result of centrifugal force and forms a microsphere. It is then collected in a cooling cham~er maintained at r: .

. W093/06208 PCT/~'S92/075X'~
2116~25 g ambient conditions or slightly lower, sized and readied or packaging.
While ro~ary disc encapsulation is known, it is not known to ma~e microsphere contained in a matrix without a surrounding shell, nor is it known to use the microsphere process or encapsulation with freeze 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 the Journal of Gas Chromotoqraphv, October, 1965, pages 345-347. In addition, a rotary disc encapsulator suitable for use in this invention is described in detail in ~nited States Letters Patent, Sparks, 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 dscribed in the parent ~hat is most preferred.
It is imp~rtant to note that rotary microsphere formation provides a distinctly different product than either conventlonal tower spray drying or microencapsulation. In conventional tower spray drying ther~ is a tendency for particles to cluster, for the coating to be uneven, and thus for the .
stability of.~he product to be significantly effected perhaps from days ~o weeks.
Microencapsulation provides a shell coatin~ around an obJect, and bacteria have proven to be too small, too hard to keep alive or provide in a ~niform size to be of practical usefulness. With microsphere formation, particularly with agen~s used in ~his invention is used, the stability of the resulting bacteria, even when subjected to some moisture and antibiotics, will be for from three to six months with the viability of the bacteria maintained in evenly distributed particles.
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^,~' W093/06208 PCT/~'S92/075X~t 2116525 lo~ -When the ~ree fatty acid microspherss of the present invention are used within the ranges hereinbefore expressed, the rotary disk, typically employing a 41'-6" rotary disc, can be run at the rate of from 20~0 rpm to 4000 rpm, prefer~bly about 2500 rpm to 3200 rpm with a feed rate of from 50 grams to 200 grams per minute. The preferred conditions pres-ently known are use of stearic acid, use o two hereinbe~ore descrlbed organisms, a four inch rotary disc, 3000 rpm and a feed rate of 100 grams per minute with a bacteria/stearic acid slurry of 35~
bacteria, 65~ stearic acid. When this is done, a product having a particle si2e 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 limit, the process of the present invention~ The examples are described in connection with Figures 1, 2 and 3. Examples 1 through 4 and Figures 1, 2, and 3 relate to the invention of my prior case. Example 5, and tables 2-10, relate to the process of this present invention for a poultry probiotic.
Example 1 Example 1 correlates with Figure 1~ It shows th~ product stability of two different strains of Enterococcus faecium with temperatures of 4C and 27C. As illustrated ln Flgure 1, it shows a stability of the encapsulated strains of En~ero-coccus faecium, with the encapsulation being by the rotary disc device using stearic acid with a level of 35~ culture weight. Conditions of microsphere ormation were as previously described herein, namely a 35/65 bacteria stearic acid slurry at a temperature of 60C, using a four inch rotary disc, operating at , WO93t0~08 PCT/~IS92/075~') - 21~65~5 3000 rpm and a feed rate of 100 grams per minute.
The spheres were formed, placed in heat sealed vapor barrier pouches and destructively sampled waekly fQr CFU determination. It can ~e seen that the product of the invention maintained ~xcellent organism colony forming unit (CFU) counts out to storage times at long as 70 days.
Exam~le 2 Example 2 is to be interpreted in connection with Figure Z. 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 consis~ed of the following:
54% five cracked corn 26~ soybean meal 2~ fish meal 1~5~ dicalcium phosphate 1~ limestone 5 . ~f% soy oil ' 12% moisture content Three antibiotics were added at the following inclusion rates by weight: deco~uinoate 6% (454 ppm), salinomycin (50 ppm) and monensin sodium (120 '! ppm)-~f~ Culture was added to the mixture at a level to deliver approximately lxlO6 CFU/gm feed. Feed was packaged in heat sealed bags and incubated at room -, temperature. Samples were taken weekly for CFU
~ determination. The graph of Figure 2 illustrates the , , excellent stabili~y.

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WO93/0620B PCT/US92/0758') Example 3 Example 3 is to be interpre~ed in conjunction with Figure 3. It shows the stability of the . .
Enterococcus faecium microspheres in eed 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 about 14%. Culture was added to a level of approxima~ely 106 CFU/gm feed and mixed. ~en pound ali~uots were stored in sealed bags at 20 C and sampled weekly for 16 weeks. The antibiotics were included in the ration at the following levels:
Bacitracin methylene disalicylate .... 50 gm/ton Carbadox ............................. 50 gm/ton Chlortetracycline ................... 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 g~/ton Furadox ................................... 10 gm/ton Table 1 is a list of the minimum times ~or a 1 ` log l~ss in colony forming units (CFU).

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W093/0620~ PCT/~;~92/075~') ~11 652a Table 1 Tlme in weeks for loss of 1 log CFU counts at 20C in 14% moisture mash feed.

Antibiotic Time of Storaqe (da~s) Control 103 Bacitracin 88 Carbadox 54 Chlortetracycline 60 Lasalocid 57 Lincomycin 75 ~eomycin 53 Oxytetracycline 59 Sulfametha~ine 62 Tylosin 52 Virginiamycin 112 : ASP250 : 67 ' ~ Furadox 53 ., ~
Example 4 . ~
In Example 4 the:s~ability of product after pelletizi~g for ~se of a chicken feed product was ~: determined. The microsphere formation conditions were as earlier described. The conditions used in this study were tha~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 (CTC
50 gm/ton~ were made with the following ingredients . and conditions.
Corn, SBM, whey, soy oil, dicalcium phosphate, limestone, tr~Ge mineral premix, vitamin premix, ~ selenium, copper sulfate. Culture was added at i~ approximately 5x105 CFUlgm feed.

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W093/062~ PCT/US92/075X'J
, - 2~ 2~ 14 -Conditioning temperature was 70C and the pellets out of the dye were 78C.
Pelle~s were stored in unsealed bags and sample~ .
weekly for CFU determination.
In each instance the pelletized product was not adversely affected ln stability by the conditions of pelletizing. In particular, the pelletized product showed stability egual to the unpellstized product.
Example 5 : Four thousand five hundred sixty, day-old Peterson x Arbor Acres broiler chicks were randomly assigned to floor pens (Table 2) with reconditioned litter and fed for 45 days. All birds dying during the first 5 days were replaced with a same-sex bird from the same shipment and same treatment. The composition of the basal starter, grower, and I withdrawal rations i5 shown in Table 3. Star~er, .31 grower f and withdrawal rations were formulated to j con~ain 1425, 1450, and I475 kcal ME/lb, respectively, wi~h 90 g/ton monesin. Starter rations ~ were fed from l to 21 days of age, grower from 21 to `~ : 42 da~s Qf age, and wi~hdrawal from 42 to 49 days of i ag~. The treatments were negative control, mash ~ (Contrbl, M); a:selected, encapsulated probiotic `; cultures:~containing EnterocQccus faecium 301, DSM No.
D5M-Nr. 4789 and Enterococcus faecium 202, DSM No.
DSM-Nr. 4788 each rotary disc fatty acid encapsulated l as described in Example 1 and each present as 50% of '. the probivtic applied at 1 x 10 CFU/g of feed, mash (prob~otic, M); negative control, pelleted (Control, ~jJ~, P); probiotic applied at 1 x 106 CFU/g mash, pelleted ` (probiotic, P) and a positive control applied at 10 `: g/ton virginiamycin, pelleted (Stafac ~ 10). The . starter ration was crumbled for the treatments that i.~ were pelle~ed. Twelve replicated pens of 35 males ., ,, , . ~

~093/~6~0X PCT/U~92/07~X"

and 35 females were used with each experimental ration.
Body weights, feed consumption, and mortality.
after the first 5 days were recorded by pen. Feed conversion, adjusted feed conversion, and body-weight adjusted feed conversion were calculat~d ~or each pen .
All data were sub;ected to analysis variance and differences were d~termined using Fisher LSD.
Prior to the study, probiotic culture concentrate was extended with calcium carbonate. The theoretical counts for probiotic, M and probiotic, P
were 1 x 108 and 2 x 109 CFU/g of product, respectively. An 11 g sample of each product was assayed in duplicate to determine actual product counts. Each sample was plated using the Pioneer 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 probiotic was uniformly distributed at appropriate levels in the feed and~hat it sur~ived pelleting.
Each ~atch was sampled at the time of bagging with 4 equally spaced samples for the mash treatments and 10 equally spaced samples for the pelleted treatments e. bags 1, 3, 5,..., 35, 37, and 39).
Alternate 1Oor pens within a treatment had non-contaminated feed sampled during weeks 1 and 4; with the remaining pens sampled on weeks 2 and 6 during the feeding study.
An equaI number of birds from each sex was sacrificed for the determination of individual ~ ~ breast, body and small intestinal weights, and small !i intestinal length. Breast yield and intestinal weight and length ratios were calculated for each bird.

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All data were subjected to a split-plot analysis o~ variance and differences were determined using contrast and estimate statements for the desired . .
e~fects.
Sixty birds per treatment were transported to a university for a sensory taste panel evaluation.
Probiotic, regardless of processing, improved (P<.05) feed conversion over the respective Control while increasin~ (P~.05) weight gain over the Control only in the mash feed (Table 4). The probiotic, P
improved tP~.05) feed conversion over Stafac ~ 10 which was similar (P>.05) to Control, P.
The product was at its desired level and strair;
composition (Table 5).
Probiotic was uniformly distributed within the feed. Probiotic, M was at its desired level while probiotic, P was 1 to 1-1/2 log higher than desired for the starter and grower rations (Table 6). The high counts for probiotic, P were a result of overengineering of the product to ensure sufficient recovery of the organisms af~er pelleting.
The floor pen samples for the probiotic, P
corresponded closely with the counts from the mixer tests ~Table:7). However, probiotic, M dropped 2 logs in weeks 4 and 6 in the grower and withdrawal mixes.
Probiotic, M increased (P<.05) both breast weight and yield over the Control, M (Ta~le 8) while probiotic, 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 probiotic, P did not show a similar magnitude in improvement in breast yield to that observed in probiotic, M. This failure may be due to improved ener~y utilization by pelleting resulting in less room for improvement.
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Pelleting increased the average bird weight by ~6 g over mash. Pro~iotic 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. Probiotic increased the average breast weight and uniformity (Figure 4) over the Control with the greatest improvement f ound in mash .
Stafac ~ 10 showed the greatest improvement in uniformity for the pelle~ed feeds.
Pelleting increased breast yeild by .53 percentage units over mash. Probiotic, M showed a .84 percentage unit incxease over Control, M which was similar in magnitude to the pelleting respoonse.
The probiotic 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). Probiotic, M had a lighter (P>.05) small intestinal weight than Con~rol, M when ~expressed as either actual weight or percentage of A~ either body or breast weights. The reduction in intestinal weight and length for probiotic 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 probiotic, P treated birds produced no off-flavor when compared ~o Stafac ~ 10 (Table 10). In ~, ~ the second trial, probiotic, P was perceived to have enh~nced the flavor of the thigh/leg when compared to Control, P. However, this enhancement of flavor was not observed in the first trial.
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PEN ASSIGMMENTS

Treatments Pen numbers -Control, P 2,6,15,17,22,26,104,10g,113,117,122,1Z6 Probiotic, P 4,8,12,16,21,28,105,106,112,118,125,130 Stafac0 10 5,7,11,18,23,27,101,107,111,116,123,129 Control, M 3,9,13,20,24,30,102,10~,114,11g,121,127 Probioti~, M 1 , 10 , 1 4 , 19 , 25 , 29 , 103 , 110 , 115 , 120 , 124 , 128 Pen size 4.2~ x 15.5~, one tube feeder, one hanglng wate~er, pine shavings on dirt/ power and evaporative eooling system and well insulated, forced hot-air heat, curtain sidewall building.

COMPOSITION OF BASAL R~TIONS

i Production Phases 3 Ingredients Starter Grower ~ Withidrawal l~ ~ Ground corn ~ 65.37 67.89 74.29 i So~bean meal 25.58 23.53 17.83 ~ ~ MFett and bone ~ieal ~ 3 6 3 32 3 00 ~ Deflourinated p~ospha~e ~95 .79 .73 Calciu~ carbonate ~ 615 631 32 Trace m~neral .05 .05 .05 Methionine 19 06 18 Vitamin premix .05 .05 Y
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, , -1~9-21~6S25 TABL~ 4 FLOOR PN PRODUCTION DA~

PolletStafac~ lnv ~
Control P 10 Control M
_ ~eight, lb. 4.79~ 4.81~4 79~ 4,54b 4.6~-~eed conv. l.B71b 1.827' l.B55~ b 1 . 917c 1,~56 Adj. feed conv.l 1.832b 1.789~07~ 1.8~7C 1.512 We ght, adj 2 l.B01b 1.755' 1.775 1.897C 1.798b Mortality, ~ 4.40 4.64 5.95 3.33 5.60 Adj feed conversion ~ Total feed/~live + dead weight).
Weiaht adj. feed conversion Y Adj. feed conversion~ weight-4.60)/6).
~b~ p< 05 i' * Inv = Invencion PRO W CT QC AND QA

Treatments ~ count ~A2count Strain ratio 1:
l Probiotic, P S.75~x 10~ 1.01 x 10~ 50:50 Probiotic,~M , : :~.54 x 107 9.60 x~10 57:43 , :
1. Qualit~ control . Quality assurance.
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-20- 2116~5 FEEDMILL MIXER IEST AND RECOVERY

Produc~tion Phases and TreatmentsMash Pellet . Recover~
~ ~ cfu/g of ~eed - % ma~;~ ~~~

Probiotic. P 2.02 x ~o6 1 67 x 10~ 98.69 Stafac~ 10 NA 6.46 x 10 Control, M Z.51 x 103 Probiotic, M 1.34 x 10 Grower Control, P NA 4.86 x 102 Probiotic, P 3. as x 106 1 . 09 X 106 91 . 62 Stafac~ 10 5.25 x 10~ 6.42 x 103 Control, M 1.00 x 102 Probiotic, M 1.48 ~ 10 Probiotic, p B 50~x 10~ 1 11 x 103 117.40 Stafac~ 10 8.80 x }03 1.79 x 10 Control, M 8.92 x 10 Probiotic, M : 1.33 x 10 Mean Control, P : 8 50 x 102 8.28 x 102 Probiotic, P 8 2~ x 1~5 9.64 x lQ6 llB.09 Stafac~ 10 2.15 x 104 9.05 x 10 -Control, M B.72 x 102 ~: Probio~ic, M ~ 1.38 x 105 Recovery calculated on logiD transformed data.
2 NA means not available.

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Weeks Treatment:s 1 2 4 6 Mean - cfu/g of leea Control 3.78 x 102 3.83 x 1028.60 x 102 2.21 x 102 4.08 x 102 Probiotic, P9.23 x 1059.37 x 1058~77 x 105 8.4~ x 105 8.96 x 105 Stafac~ 10 8.73 x 102 1.29 a~ 1026.46 x 102 8.63 x 102 8.89 x 10 Control, M 3 46 x 102 1.26 x 1022.79 x 103 2.00 x 102 5.08 x 10 Probiotic, M 1 43 x 1051.25 x 1051.75 x 103 1.00 x 103 2.32 x 104 .

BRE:AST YIELD EVALW~TION

PIel * -- stafac~ Inv *
ontrol P 10 Control M

Body weight, g ~ ~ ~ 2240 7 2230 1 2195 9 2143 8 2149 9 ~ ~ Br~ yield, % of ; ~ 10 . 68A 10 . 58~ 9, 93b 10 . 67A

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2116~
TABLE ~
IN~STINAL t~EIGH~ AND LENGTH

Pelle~t Stafac~
Control P 10 Control _ . _ Body weight, g 2240.7 2230.1 2195.9 2143.~2149.9 Breast weight, g 234.4~ 239.6~ 232.0~ 213.3b 229.6' SI weight~ g 92.6 3.3 93.4 91.4 .87.4 SI, g/in 71 31 715 23 71 22 71 107~ 16 SI we ght~ g/100 g4.17 4.18 4.27 4.29 4.08 SI length, in/loo g body weight 3.47 3.40 3.53 3.61 3.53 SIbreagt weight 40.19 39.70 40.97 43.96 38.69 SI length, ig~lt 33.41~ 32.27~ 33.72~ 36.89b 33.41-`!
J, ab P~ . 05 3; * Inv = Invention ~ ~ SX ~ Small Inte~ine i~' - TABLE lO
~ ~ TASTE PAN~ EV~IL~TION
s - Group ~ - umbIr of correct identifications Tissue Co~parison : Trla Tria 2 C ne gh,/leg Stafac~:10 YS. Co~trol, P 6 3 9 Stafac0 lC vs. XINOC, P 3 4 7 - Probiotic, P vs. Control, P 2 8* 10 : Breast: Stafac~D lO vs. Controlr P 2 6 8 - ~ Staac0 lO v~. XINOC, P 1 3 4 P~obiotic, P vs~. Control, P 5 4 9 The evaluators were able to detect the odd sample a statistically signlficant (P<.05) n~ber of times.
he number of correct identifications o~ the Oda siæmple required for significance at the 5~ level was 7 for n=10 and 11 for n=20.

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Claims

What is claimed is:

1.
A method of growth promotion of poultry, comprising:
adding to a normal poultry feed ration a small but growth promotion effective amount of probiotic consisting essentially of viable, stable, dried fatty acid microspheres of Enterococcus faecium 301, ATCC No. , and viable, stable, dried fatty acid microspheres of Enterococcus faecium 202, ATCC No.

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

3.
The method of claim 2 wherein the probiotic 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 each of said streptococci are present in about equal amounts.

7.
The method of claim 1 wherein the amount of probiotic added to the feed ration is from about 0.5 pounds to about 2.0 lbs./ton of feed.

8.
The method of claim 7 wherein the amount of probiotic is from about 0.8 lbs. to about 1.2 lbs./ton of feed.

9.
The method of claim 7 wherein the organism count of the probiotic is from about 1 x 105 CFU/gm to about 2 x 108 CFU/gm.

10.
The method of claim 9 wherein the organism count of the probiotic is about 1 x 105 CFU/gm.

11.
A probiotic composition for growth enhancement of poultry consisting essentially of viable, stable, dried fatty acid microspheres of Enterococcus faecium 301, and viable, stable, dried fatty acid microspheres of Enterococcus faecium 202.

12.
A probiotic of claim 11 which has from about 30 to about 20% of one of said streptococci with the balance being the other.

13.
The probiotic of claim 12 wherein the free fatty acid is a C12 to C24 free fatty acid.

14.
The probiotic of claim 13 wherein the free fatty acid stearic acid.

15.
The probiotic of claim 14 wherein the streptococci organisms are present in about equal amounts.