WO1993006208A1

WO1993006208A1 - Fatty acid microencapsulated enterococcus for use with poultry

Info

Publication number: WO1993006208A1
Application number: PCT/US1992/007589
Authority: WO
Inventors: William M. Rutherford; Jack E. Allen; Scott M. Dennis; Mark A. Hinds; Gregory R. Dana
Original assignee: Pioneer Hi-Bred International, Inc.
Priority date: 1991-09-20
Filing date: 1992-09-09
Publication date: 1993-04-01
Also published as: MX9205329A; HUT67466A; SK32494A3; HU9400794D0; SK278992B6; CZ59594A3; BR9206505A; BG62175B1; CA2116525A1; CZ280601B6; JPH06511148A; EP0604543A1; RU2093571C1; RO113477B1; BG98665A; EP0604543A4

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

FATTY ACID MICROENCAPSULATED ENTEROCOCCUS

FOR USE WITH POULTRY

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 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 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 probiotics which contain desirable

bacteria. Probiotics, however, do have some difficulty in maintaining a stable 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. 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 activity of the probiotic is stopped. In other instances, the addition of antibiotics to the probiotic containing 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, there is a significant problem of long term storage stability for

probiotics.

In another environment, where the probiotic is added to, for example chicken feed, 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

inactivating bacteria before they really reach the intestine. Thus, there is a continuing need for probiotics 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.

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 course, 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 birds, 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.

It is a primary objective of the present

invention to provide a poultry probiotic which contains no antibiotics and contains only fatty acid microencapsulated naturally occurring organisms.

It is another primary objective of the present invention to provide a probiotic which contains two organisms, namely Enterococcus faecium 301, DSM No. DSM-Nr. 4789, and Enterococcus faecium 202, DSM No. DSM-Nr. 4788. DSM is a Bacterial Culture collection in Germany. DMS stands for Deutsche Sammlung von Mikroorganismen located in Braunschweig, West

Germany. These 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 probiotic which, for poultry, provides increased rate of weight 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 size.

An even further primary objective of the present invention is to provide probiotics 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 probiotic which has stability at levels within the range 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 of the dried bacteria which provides having uniform size.

Another objective 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 DESCRIPTION OF THE DRAWINGS

Figures 1, 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 probiotic composition of the present invention.

Figure 5 is a graph showing body weight

Figures 4 and 5 show a control, use of an

antibiotic and use of the probiotic of the present invention. SUMMARY OF THE INVENTION

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, fatty acid microspheres of

Enterococcus faecium 301, DSM No. DSM-Nr. 4789, and dried fatty acid microepheres of Enterococcus 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 can be accomplished by adding to normal poultry feed rations, a certain amount of fatty acid microspheres of Enterococcus faecium 301, DSM No. DSM-Nr. 4789, and a certain amount of fatty acid microspheres of Enterococcus faecium 202, DSM No. DSM-Nr. 4788. A fatty acid employed may be any 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 other.

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 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.

The amount 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 feed, and typically at about 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 10⁶ CFU/gm to about 2 x 10⁹CFU/gm, but is preferably at about 2 x

10⁸ CFU/gm.

When the probiotic as previously described is free choice fed in poultry feed ration, the

combination of two strains of organisms herein mentioned, behave as a growth promoter. Growth promoters now used include antibotics 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 probiotic is 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 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 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. This process if described in the parent application of the coinventor 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. The

combination of the factors provides for a highly stable probiotic 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 dosing with the bacterial treatment.

The preferred encapsulating agent is a C₁₂ to C₂₄ free fatty acid. While mixtures of fatty acids 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 speaking, it is important that the fatty acid have a melting point less than 75°C, preferably within the range of 40°C to 75°C. 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 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.

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 bacteria and fatty acid components are thoroughly mixed with the mixture being added at a uniform rate 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 chamber maintained at ambient conditions or slightly lower, sized and readied for packaging.

While rotary disc encapsulation is known, it is not known to make 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 Chromotography.

October, 1965, pages 345-347. In addition, a rotary disc encapsulator suitable for use in this invention is described in detail in United 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 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.

Microencapsulation 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 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. 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 grams to 200 grams per minute. The preferred conditions presently known are use of stearic acid, use of two hereinbefore described 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 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 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 the product stability of two different strains of

Enterococcus faecium with temperatures of 4°C and 27°C. As illustrated in Figure 1, it shows a

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 stearic acid slurry at a temperature of 60°C, using a four inch rotary disc, operating at 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 weekly for CFU determination. 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 interpreted 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 following:

54% five cracked corn

26% soybean meal

2% fish meal

1.5% dicalcium phosphate

1% limestone

5.5% soy oil

12% moisture content

Three antibiotics were added at the following

inclusion rates by weight: decoquinoate 6% (454 ppm), salinomycin (50 ppm ) and monensin sodium (120 ppm).

Culture was added to the mixture at a level to deliver approximately 1x10 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 stability. Example 3

Example 3 is to be interpreted in conjunction with Figure 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 about 14%. Culture was added to a level of approximately 10 CFU/gm feed and mixed. Ten pound aliquots 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 gm/ton

Furadox.......................................................... 10 gm/ton

Table 1 is a list of the minimum times for a 1og loss in colony forming units (CFU). Table 1

Time in weeks for loss of 1 log CFU counts

at 20C in 14% moisture mash feed.

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 (CTC

50 gm/ton) were made with the following ingredients and conditions.

Corn, SBM, whey, soy oil, dicalcium phosphate, limestone, trace mineral premix, vitamin premix, selenium, copper sulfate. Culture was added at approximately 5x10⁵ CFU/gm feed. Conditioning temperature was 70°C and the pellets out of the dye were 78ºC.

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.

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

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 1 to 21 days of age, 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 probiotic cultures containing Enterococcus faecium 301, DSM No.

DSM-Nr. 4789 and Enterococcus faecium 202, DSM No.

DSM-Nr. 4788 each rotary disc fatty acid encapsulated as described in Example 1 and each present as 50% of the probiotic applied at 1 x 10⁵ CFU/g of feed, mash

(probiotic, M); negative control, pelleted (Control,

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 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 days were recorded by pen. Feed conversion, adjusted feed conversion, and body-weight adjusted 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, probiotic culture

concentrate was extended with calcium carbonate. The theoretical counts for probiotic, M and probiotic, P were 1 x 10 8 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 that it survived pelleting. Each batch 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 (i.e. bags 1, 3, 5,..., 35, 37, and 39).

Alternate floor 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 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 intestinal weight and length ratios were calculated for each bird. All data were subjected to a split-plot analysis of variance and differences were determined using contrast and estimate statements for the desired effects.

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 increasing (P<.05) weight gain over the Control only in the mash feed (Table 4). The probiotic, 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).

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 after 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 (Table 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 energy utilization by pelleting resulting in less room for improvement. Pelleting increased the average bird weight by 96 g over mash. Probiotic 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 found in mash. Stafac® 10 showed the greatest improvement in uniformity for the pelleted feeds.

Pelleting increased breast yeild by .53

percentage units over mash. Probiotic, M showed a .84 percentage unit increase 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 Control, M when

expressed as either actual weight or percentage of 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 to Stafac® 10 (Table 10). In the second trial, probiotic, P was perceived to have enhanced the flavor of the thigh/leg when compared to Control, P. However, this enhancement of flavor was not observed in the first trial.

* Pen size 4.2' x 15.5', one tube feeder, one hanging waterer, pine shavings on dirt, power and evaporative cooling system and well insulated, forced hot-air heat, curtain sidewall building.

1 Adj. feed conversion = Total feed/(live + dead weight).

2 Weight acj. feed conversion = Adj. feed conversion-( (weiαht-4.60)/6). a^bc P<.05.

* lnv = invention

1. Quality control

2. Quality assurance,

* The evaluators were able to detect the odd sample a statistically significant (P<.05) number of times.

1 The number of correct identifications of the odd sample required for significance at the 5% level was 7 for n=10 and 11 for n=20.

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 C_{2 4} 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./tonn 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 C_{2 4} 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.