Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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34. EP1482811 - 08.12.2004
PROBIOTIC DELIVERY SYSTEM

URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP1482811


Inventor(s): CAVADINI CHRISTOPH (CH); UBBINK JOHAN BERNARD (CH); SCHAER-ZAMMARETTI PRISCA (CH)
Applicant(s): NESTLE SA (CH)
IP Class 4 Digits: A61K; A23K; C12N; A23L
IP Class: A23K1/00; C12N1/04; A61K35/74; A23L1/03
Application Number: EP20030711996 (20030312)
Priority Number: WO2003EP02597 (20030312); EP20020005607 (20020312); EP20030711996 (20030312)
Family: EP1482811
35. EP1495109 - 16.10.2003
METHOD OF PREPARING A PROBIOTIC PREPARATION

URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP1495109


Inventor(s): PEKELHARING PETER REMCO (NL); VAN LEEUWEN ABRAHAM BENJAMIN J (NL)
Applicant(s): WINCLOVE BIO IND B V (NL); PEKELHARING PETER REMCO (NL); VAN LEEUWEN ABRAHAM BENJAMIN J (NL)
IP Class 4 Digits: A61K; C12N; A23C
IP Class: C12N1/04; C12N1/20; A61K35/74; A23C9/123
E Class: A61K35/74; C12N1/20
Application Number: WO2003NL00255 (20030404)
Priority Number: NL20021020301 (20020404)
Family: EP1495109
Equivalent: AU2003235420; NL1020301C
Cited Document(s): GB2193875; WO9729640; WO9827824; FR2750298; EP1177794; US5902578; EP0861905; EP1243181
Abstract:

THE INVENTION RELATES TO A METHOD OF MANUFACTURING A PROBIOTICALLY ACTIVE PREPARATION. THE METHOD ACCORDING TO THE INVENTION IS CHARACTERISED IN THAT A COMPOSITION COMPRISING A PROBIOTIC MICROORGANISM AND A METABOLISABLE SUBSTRATE IS CONTACTED WITH A NON-STERILE AQUEOUS LIQUID AND IS INCUBATED FOR AT LEAST FIVE MINUTES. THIS ALLOWS A USER TO TAKE THE PREPARATION WHEN THE MICROORGANISM IS IN A DIVIDING PHASE.Description:

Method of preparing a probiotic preparation

The present invention relates to a method of manu- facturing a probiotically active preparation.


Such preparations and methods of their manufacture are generally known, and often comprise culturing a probiotic microorganism on a medium containing a liquid substrate, such as a dairy product. The preparations are taken for the pre- vention of, or to promote the recovery from intestinal com- plaints.
Due to the central and industrial-scale production, the probiotic microorganism in the preparation is not quite effective when consumed by the user.
It is the object of the present invention to provide a method by which a probiotic preparation can be manufactured with a more effectively working probiotic microorganism.
To this end the method according to the present in- vention is characterised in that a composition comprising a probiotic microorganism and a metabolisable substrate is con- tacted with a non-sterile aqueous liquid and is incubated for at least five minutes.
Incubation of the probiotic microorganism in the presence of substrate induces the microorganism, which will usually be dormant, to enter a dividing phase. The composi- tion can subsequently be taken by a user and the microorgan- isms will be better able to carry out their favourable activ- ity in the gastrointestinal tract. Duration of incubation is suitably at least 5 minutes, preferably at least 20 minutes and more preferably at least 1.5 hours. In other words, incu- bation takes place as long as is necessary for activation af- . ter dormancy and preferably for one or several divisions of the probiotic microorganism to take place. The method accord- ing to the present invention also makes it possible to use water of a biologically dubious quality. Aided by the lower- ing of the pH caused by fermentation, the probiotic composi- tion inhibits, inactivates, or even kills possible pathogenic microorganisms present in the water. In practice, incubation takes place under non-sterile conditions, such as in a non-

sterile container, and in a non-sterile aqueous liquid. After incubation the preparation is ready for consumption and, in contrast with known commercial preparations that are packaged after manufacture, this preparation is in practice not pack- aged. "Substrate"is understood to be a substrate that can be metabolised by the probiotic microorganism and comprises, for example, maltodextrin, fructo-oligosaccharides or the like.


Optionally traces of minerals such as potassium, manganese and magnesium may also be present to promote the growth of the probiotic microorganism. When referring in the present application to an aqueous liquid, this is understood to be any liquid containing sufficient water to enable the probi- otic microorganism to divide. If the composition comprises several components, among which in particular one or several probiotic microorganisms and substrate, these may be packaged separately in the form of a kit. A first package may, for ex- ample, comprise one or several probiotic microorganisms to- gether with an inert carrier, and the second packet may com- prise the substrate. Both packets are added to the aqueous liquid. However, preferably the composition comprises a com- bination, such as a mixture, of substrate and probiotic mi- croorganism, which combination therefore requires only one single packet. Preferably, the composition is packaged as "unit doses", where the packet such as a sachet, contains an amount of composition to be contacted with a previously de- termined amount of aqueous liquid. In this way a dose of the preparation ready for consumption is produced. As mentioned above, the substrate and the microorganism for the composi- tion may be packaged separately, that is to say each may be packaged as a unit dose. The probiotic preparation according . to the invention is usually a drink. When using the term "non-sterile", in the present invention, the probiotic micro- organisms of this preparation are left out of consideration.
The non-sterile aqueous liquid is preferably water, in particular water that of itself does not contain any organic carbon source as substrate, such as mineral water, tap water or natural water.

Thanks to the method according to the invention, the non-sterile aqueous liquid can be made better suitable and can even be made safe for consumption. This can also be done indirectly, if the water is used to wash fresh vegetables and fruit, which in some countries holds an increased risk of causing intestinal disorders, such as diarrhoea. The method according to the invention has been shown to reduce the bac- terial count of potentially harmful microorganisms such as E. coli. For this particular application the incubation period is preferably longer than 6, more particularly longer than 10 hours. For this application it is not important whether the probiotic microorganisms have after incubation returned to a stationary, dormant condition, though in accordance with the invention, it is still preferred for them not to be in a dor- mant condition.


According to an important embodiment, the composition comprises a growth indicator and incubation lasts until the growth indicator shows a change in colour.
In this way a variety of factors can be taken into ac- count such as the occasionally changing incubation condi- tions, viability of the probiotic microorganism, etc. A growth indicator shows at least that the microorganism has come out of dormancy and when consumed is thus able to imme- diately produce a favourable probiotic effect. In the case of reducing the bacterial count of possible pathogenic microor- ganisms it is preferred to use a growth indicator that exhib- its a delayed change.
The growth indicator used in a preferred embodiment is a pH indicator.
In this way a suitable length of incubation is shown.
This is of particular importance in conditions where the in- cubation temperature cannot be adjusted or regulated. The in- dicator shows when the microorganisms have divided suffi- ciently often or the pH of the water is sufficiently lowered to kill possibly present pathogenic microorganisms. According to an alternative embodiment, the growth indicator used may be a substrate that through cleavage produces a dye, as is well known in the field of microbiology. An example of such a

growth indicator is an indicator substrate for beta- galactosidase, such as orthonitrophenyl-b-D- galactopyranoside. Although this works well and will be pre- ferred in buffered solutions, from an economical viewpoint and for many practical applications it will be preferred to use a pH indicator in non-buffered or only slightly buffered solutions such as water. The term buffered in this case re- fers to buffered in the pH range that in the absence of the buffering substance can be attained by the probiotic microor- ganism prior to the formation of acid and after the formation of acid.


Preferably a pH indicator is used having a point of change at pH = 5 or lower, more preferably at a pH between 3.5 and 4.5.
At such a pH, effective killing or inactivation of pos- sibly present pathogenic microorganisms is achieved. A change to such a pH also provides a good indication of sufficient division of the probiotic microorganism. When choosing an in- dicator around pH 4 or lower, the change will take place later, which is favourable for an application such as the re- duction of the bacterial count of possibly pathogenic micro- organisms.
It is preferred to use as the probiotic microorganism at least one probiotic microorganism chosen from Lactobacillus, Lactococcus, Propionibacterium, Pediococcus, Bifidobacterium, Enterococcus, and Streptococcus.
Microorganisms such as, for example, Lactobacillus aci- dophilus, Lactobacillus casei, Lactobacillus salivarius, Lac- tobacillus plantarum, Lactococcus lactis, Propionibacterium freudenreichii, Pediococcus acidilactici, Bifidobacterium lactis, Enterococcus faecium, and Streptococcus lactis, make it possible to produce an effective probiotic preparation. It is, of course, possible to use a mixture of more than one probiotic microorganism, taking care that each probiotic mi- croorganism has a substrate at its disposal to enable it to bring the microorganism into a dividing (log-) phase.
According to a preferred embodiment the composition is a dry composition.

In contrast with many of the usual commercially avail- able probiotic preparations, such a composition can be stored for a long time, and takes up little space. In addition the shelf life is considerably extended without jeopardising the effectiveness. This is especially the case in the presence of a growth indicator. Within the scope of the present inven- tion, "dry"means in the absence of so much water as to ren- der the microorganism dormant. The manufacture of dry compo- sitions comprising microorganisms is well-known in the art.


It may be realised, for example, by means of spray-drying or lyophilisation. Such a composition requires no cool storage.
The metabolisable substrate may be mixed with the microorgan- ism either prior to but preferably subsequent to drying the microorganism. The composition may comprise a carrier such as cornstarch.
The invention also relates to a probiotically active composition, which is characterised in that the composition comprises a probiotic microorganism, a metabolisable sub- strate for the microorganism and a growth indicator, and op- tionally a pharmaceutically acceptable carrier or excipient.
Preferably the composition is a dry composition.
In contrast with known probiotic compositions currently on the market, such a composition has a very long shelf life.
It will be obvious to the ordinary person skilled in the art that the invention may be applied in a variety of ways.
For example, it is possible to carry a preparation made up with water in a container such as flat flask against the body to provide an environment of elevated temperature enabling the probiotic microorganism to grow more quickly and allowing the ready preparation to be used sooner.
The invention will now be elucidated with reference to the following examples.
Example 1

As model pathogen use was made of E. coli ATCC 25922 cultured in Brain Heart Infusion (BHI) -broth by incubating for 18 hours at 370C. This provided mature cultures comprised of approximately 108 colony-forming units (cfu) per ml. From


this solution a dilution series was derived in a sterile pep- tone physiological (pfz) ) solution.


Subsequently various samples (100 ml) of Amsterdam tap water were inoculated with different E. coli ATCC 25922 con- centrations, to wit:

Sample Initial conc. E. coli

(cfu/ml)

A 3. 8*102

B 3. 8*103

C 3. 8*104

D 3. 8*105

E 3. 8*106

F (control) 4. 0*106

G (control) 4. 0*104

After that a known'concentration (4.8 * 107 cfu/ml) of the probiotic composition was added, consisting of 1% Lacto- bacilli (L. plantarum, L. casei, L. acidophilus and L. sali- varius at approximately equal quantities), 20% maltodextrin, 5% dextrose, 5% mineral mix (equal quantities of potassium chloride, magnesium sulphate and manganese sulphate), 64% cornstarch (carrier) and 5% caranthopowder as a colour indi- cator.
In the course of the experiment all the water samples were left at 37 C (without shaking).
After 6 and 24 hours the bacterial count of E. coli ATCC 25922 was determined. Using sterile pfz decimal dilutions of the samples were made, which were subsequently incubated on media suitable for the respective microorganism. An E. coli test was carried out using Eosine Methylene Blue agar (EMB, Petri dishes). After 24 hours incubation (aerobic) at 37 C these dishes were evaluated (counted). The test for the Lac- tobacilli was performed using Man, Rogosa, Sharp Agar (MRSA, Petri dishes). These plates were evaluated after 48 incuba- tion (anaerobic) at 37 C.

In addition the pH was measured at t=0, t=6 and t=24 hours.


Control (samples F and G):

As control experiment, no probiotic bacteria were added to two different starting concentrations of E. coli (106 and 104, control F and G, respectively) whereas the other compo- nents of the composition such as the nutrients, were added.


In order to determine the influence of acid production only, the pH was adjusted by acidifying (0.1 M HC1) to the same pH that was measured in the experiment with the probiotic prepa- ration.
At different points in time a sample was taken to deter- mine the bacterial count of E. coli. At t=6 hours the bacte- rial count of the probiotic bacteria of experiment A was de- termined.
Results

The results of the study are shown in the tables and graph below.


Table 1. Total bacterial count of E. coli ATCC 25922 present in the various water samples at three points in time (expressed in cfu/ml).

sample Time (hours)

0 6 24

A 3.8*102 5.0*102 0



B 3. 8*103 1. 5*104 1

C 3.8*104 6.2*105 6.0*102

D 3.8*105 4.8*107 2.8*103

E 3. 8*106 1 O*108 6. 0*102

F (control) 4. 0*106 6. 0*107 8 0*107

G (control) 4. 0*104 2. 2*104 1. 0*107


For the probiotic microorganisms relating to experiment A and 6 hours, an increase to 1.1 x 108 cfu/ml was measured.
The pH values were 6.3, 4.1 and 3.7 on t=0, t=6 and t=24, re- spectively. After 6 hours a colour change was observed from purple-pink to deep pink.
Conclusion

The probiotic preparation inhibits the growth of E. coli ATCC 25922 in water. Incubating an initial concentration of 3. 8*102 cfu/ml of E. coli for 24 hours with the probiotic preparation, even resulted in no E. coli being detected in the water. The two control experiments in which the pH is ad- justed but no probiotic bacteria are added show that E. coli ATCC 25922 is not inhibited or that there is even some growth. This shows that the inhibition of E. coli ATCC 25922 is not or not solely caused by the pH drop but also by an- other, probiotic microorganism-dependant factor.


Example 2

In an experiment similar to that of Example 1 the influ- ence was studied of a probiotic preparation on a number of pathogens present (through addition) in both tap water from Wageningen (town in the Netherlands) and demineralised water.


The probiotic composition was the same as in Example 1.
Material and methods

Micro-organisms

The following three pathogens were used in the study:

1. Shigella flexneri (strain collection University of

Wageningen, the Netherlands) ;

2. Salmonella typhimurium (strain collection Univer- sity of Wageningen, the Netherlands);

3. Escherichia coli (strain H03-50 from the RIVM,

Lelystad, the Netherlands, isolated from faeces of a patient suffering from (bloody) diarrhoea).


For use the strains were cultured in Brain Heart Infu- sion (BHI) -broth by incubation for 18 hours at 370C. In this

way mature cultures were obtained of approximately 109 colony- forming units (cfu) per ml.


Water

Two kinds of water were used in this study: - tap water (tap water-suitable for consumption); - demineralised water (demi-water).


Measuring flasks were filled with water (100 ml water per flask) and sterilised for 15 minutes at 121 C. Thus the water will be contaminated with the test pathogens only.
Experimental set-up

To the various measures of water, with the exception of the blank series, 6.0 grams of the probiotic preparation (initial concentration 3 * 107 cfu/ml) were added. These sam- ples were subsequently inoculated with the pathogenic bacte- ria (1 type of bacteria per 100 ml water) in different ini- tial concentrations (104 cfu/ml and 106 cfu/ml). The 106 cfu/ml water was obtained by using 0.1 ml of a mature culture to aseptically inoculate 100 ml of water in a measuring flask. To obtain an initial concentration of 104 cfu/ml of wa- ter, a mature culture was (decimally) diluted with sterile peptone physiological (pfz) -solution, after which 0.1 ml of a suitable dilution was used to inoculate 100 ml water in a measuring flasks.


At t=0 (after addition of probiotica and inoculation with a bacterium), t=6, t=11 and t=24 hours samples were taken to be examined for the number of pathogens. Using ster- ile pfz decimal dilutions of the samples were made which were subsequently examined (in duplicate) in a manner known in it- self, with the aid of suitable media. Shigella and Salmonella were tested for with Violet Red Bile Glucose (VRBG) agar (cast plates), E. coli was tested for with Violet Red Bile Lactose (VRBL) agar (cast plates). The plates were assessed (counted) after 24 hours incubation at 370C. As control, 0.1 ml of a suitable dilution of the samples was spread on the selective medium Xylose Lysine Dextrose (XLD) agar to deter-

mine the typical colony appearance of Salmonella and Shigella (in order to exclude contamination).


In the course of the experiment all the water samples were set aside at 37OC (without shaking).
Results

The results of the study are presented in Table 2 below.


Once again a colour change was observed, indicating that the probiotic microorganisms were active.
Table 2. Numbers of pathogenic bacteria present in the various water samples at four points in time (expressed as log cfu/ml).
Samples t=0 t=6 t=11 t=24 Samples t=0 t=6 t=11 t=24 in demi-In tap water water

Shigella 3.8 3.5 3.2 < 0.5 Shigella 3.7 1.5 < 0.5 < 0.5

Shigella 5.4 5.5 5.5 < 0.5 Shigella 6.0 3.0 < 0.5 < 0.5

Shigella 6.0 4.8 4.6 Shigella 5.9 3.3 0.7 (blank) 4. 4 (blank) 2.9

Salmonella 3.9 1.6 1.0 Salmo-4. 0 3.2 < 0.5 < 0.5

0.9 nella

Salmonella 6.0 4.6 2.2 < 0.5 Salmo-6. 2 5.2 < 0.5 nella 2.9

Salmonella 5. 9 6.0 6.1 Salmo-6. 0 3.9 3.9 (blank) 6.0 nella 4. 0 (blank)

E. coli 3.9 2.1 2.3 < 0. 5 E. coli 3.7 3.0 0.9 < 0.5

E. coli 6.1 5.4 5.2 E. coli 5.9 5.3 2.0 < 0.5

0.5

E. coli 6.0 5.7 5.8 E. coli 6.1 4.6 4.4 3.7 (blank) 5. 5 (blank)



Conclusion

The addition of the probiotic preparation to both tap water and demi-water, in which pathogenic bacteria are present, was shown to have an inactivating effect on the pathogens. After 24 hours the pathogens are no longer detect- able in tap water. At that moment low numbers of the bacteria


Salmonella and E. coli are still detectable in demi-water.Claims:

CLAIMS

1. A method of manufacturing a probiotically active preparation, characterised in that a composition comprising a probiotic microorganism and a metabolisable substrate is con- tacted with a non-sterile aqueous liquid and is incubated for at least five minutes.


2. A method according to claim 1, characterised in that the non-sterile aqueous liquid is water.
3. A method according to claim 1 or 2, characterised in that the composition comprises a growth indicator and in- cubation lasts until the growth indicator shows a change in colour.
4. A method according to claim 3, characterised in that the growth indicator used is a pH indicator.
5. A method according to claim 4, characterised in that a pH indicator is used having a turning point at pH = 5 or lower, preferably at a pH between 3.5 and 4.5.
6. A method according to one of the preceding claims, characterised in that as the probiotic microorganism at least one probiotic microorganism chosen from Lactobacil- lus, Lactococcus, Propionibacterium, Pediococcus, Bifidobac- terium, Enterococcus, and Streptococcus is used.
7. A method according to one of the preceding claims, characterised in that the composition is a dry compo- sition.
8. A probiotically active composition, characterised in that the composition comprises a probiotic microorganism, a metabolisable substrate for the microorganism and a growth indicator, and optionally a pharmaceutically acceptable car- rier or excipient.
9. A composition according to claim 8, characterised in that it is a dry composition.

36. EP1510135 - 02.03.2005
A FARM ANIMAL FEED PRODUCT WITH PROBIOTIC ENTEROCOCCUS BACTERIA

URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP1510135


Inventor(s): LEEDLE JANE A Z (US); JOHNSON STEVEN C (US); KAUTZ WILLIAM P (US); LECHTENBERG KELLY F (US)
Applicant(s): HANSENS LAB (DK)
IP Class 4 Digits: A23K
IP Class: A23K1/00; A23K1/18
E Class: A23K1/00C2B; A23K1/18K
Application Number: EP20030077658 (20030826)
Priority Number: EP20030077658 (20030826)
Family: EP1510135
Cited Document(s): WO8911858; WO9400019; US2001014322; US6503544; EP0856259; GB2312676; JP2002058432
Abstract:

A FARM ANIMAL FEED PRODUCT COMPRISING PROBIOTIC ENTEROCOCCUS BACTERIA AND THE USE OF THIS PRODUCT TO REDUCE THE NUMBER OF PATHOGENIC ESCHERICHIA COLI O157:H7 CELLS IN FEED FARM ANIMALS SUCH AS CATTLE.Description:


FIELD OF THE INVENTION


[0001] The present invention relates to a farm animal feed product comprising probiotic Enterococcus bacteria and the use of this product to reduce the number of pathogenic Escherichia coli O157:H7 cells in feed farm animals such as cattle.

DESCRIPTION OF THE BACKGROUND

[0002] The animal feed industry, such as the beef cattle industry, is experiencing challenges like never before and one of the most critical challenges to the industry is food safety. The consumer and governmental agencies are requiring that e.g. beef sold in restaurants, grocery stores and meat markets be as safe and pathogen free as possible. Meat packing companies are looking to the feed yards and the cattle producers to implement strategies to help achieve this goal.


[0003] The demand for food industry control of potentially contaminating pathogens starts, as noted, at the consumer's level, who are stating, through their buying patterns at the meat case, that they need a product in which they can have confidence. Subsequently retailers look to wholesalers and the packing companies. The packers are looking to the feed yards and e.g. the cattle producers to take the necessary steps to help reduce this problem by adopting safety standards and procedures at all points along the production chain. This issue is key and it will take adjustments of management procedures by all entities involved in beef production to address this issue.
[0004] It has been well documented through scientific and medical research that the predominant organism at the root of food safety issues is Escherichia Coli (E. coli) 0157: H7, otherwise known as enterohaemorrharic E. coli microorganism. E. coli O157:H7 is one of hundreds of strains of the bacterium. Although most strains are harmless and live in the intestines of healthy humans and animals, this strain produces a powerful toxin and can cause severe illness. It also possesses other significant attributes, which contribute to its ability to cause disease. One of the more notable of its characteristics is the size of the infectious dose, which is incredibly small in comparison with those for most other food-borne pathogens. Figures as low as two bacteria per 25g food have been quoted capable of creating a disease condition.
[0005] The strain E. coli O157:H7 was first recognized as a cause of illness in 1982 during an outbreak of

severe bloody diarrhea; the outbreak was traced to contaminate hamburgers. Since then, most infections have resulted from eating undercooked ground beef. The combination of letters and numbers in the name of the bacterium refers to the specific markers found on its surface and distinguishes it from other types of E. coli. Another pathogen of concern includes strains of Salmonella, with both E. coli and Salmonella commonly existing in the gastrointestinal tracts of cattle. These organisms are endemic and commonly found in virtually all phases of production. While they may not cause a problem in the host animal they can cause illness and even death in humans. Cattle become "infected" with this organism through exposure in their natural environment. After the organism is ingested it travels to the intestine where it adheres to the tract lining. Meat is "contaminated" by the organism during the slaughtering and processing stages when intestinal contents can come in contact with other meat surfaces and subsequently become mixed with ground beef.


[0006] In humans, an E. coli infection can lead to bloody diarrhea and even kidney failure. In some persons, particularly children under 5 years of age and the elderly, the infection can also cause a complication called hemolytic uremic syndrome, in which the red blood cells are destroyed and the kidneys fail. About two to seven percent of infections lead to this complication. In the United States, hemolytic uremic syndrome is the principal cause of acute kidney failure in children, and most cases of hemolytic uremic syndrome are caused by the strain E. coli O157:H7.
[0007] Most illness have been associated with eating undercooked, contaminated ground beef. In addition, however, person-to-person contact in families and childcare centers is also an important mode of

transmission. Infection can also occur after drinking raw milk and after swimming in or drinking sewage-contaminated water. As an example of this, the USDA Food Safety and Inspection Service



(FSIS) has estimated that consumption of meat contaminated with pathogenic bacteria annually results in thousands of deaths and millions of illnesses in the U. S. alone. The government estimates the annual losses in production and medical costs may reach as high as DOLLAR 35 billion. The problem is well documented and identified.
[0008] Having recognized this problem and a need to solve it or at least diminish it, recent proactive efforts have been shownby the industr. The proactive efforts exerted by e.g. the US beef industry have resulted in recommendations of expanded research and accelerated use of intervening methodologies by industry leaders. Control and treatment techniques such as the irradiation of beef products post slaughter, use of new vaccines in cattle and direct feeding of certain additives are all under serious investigation and consideration as contributing solutions. Of these, the use of feed additives has gained significant interest, largely due to simplicity of administration.
[0009] One particular group of feed additives showing significant promise in this area is probiotic or Direct-Fed Microbial (DFM) products. The use of DFM's has grown significantly over recent years largely as a means of enhancing the health and performance of the animal. The use of bacterial-based DFM's in ruminant diets for specific applications has become widely recognized. Products of this nature often contain lactobacilli with Lactobacillus acidophilus being one of the most common.
[0010] Most bacterial-based DFM's are beneficial because they have effects in the lower gut and not in the rumen. For example, Lactobacillus acidophilus produces lactic acid, which may lower the pH in small intestines to levels that inhibit the growth of pathogenic microbes, one of the reasons for the current interest. Early research with DFM in ruminants first involved applications for young calves fed milk, calves being weaned, or cattle being shipped. These animals, in many cases are highly stressed or had a microbial gut ecosystem that was not fully mature. Young cattle have immature digestive tracts that are obviously more prone to upset by pathogenic bacteria. Cattle that are shipped are often on limited feed and water for prolonged periods of time during transit. During these periods microbial populations may decrease in numbers thus resulting in digestive tracts that are in less than optimal condition. Large doses of beneficial organisms were thought to re-colonize a stressed intestinal environment and return gut function to normal.
[0011] The American Meat Institute (AMI) Foundation published in 2002 a result of a research study that was done by Mindy Brashears and Michael Galyean of Texas Tech University. The study demonstrated that the feeding of two different Lactobacillus acidophilus bacteria strains gave a significant reduction (P<.05) in the incidence of E. Coli O157:H7 in the feces of finishing cattle. The experimental design of the study was:

Control - Cattles feed with a standard diet,

NP 747 - Cattles feed with a standard diet with 1 x 10CFU Lactobacillus acidophilus strain NPC 747 mixed in water and added to the diet at the time of feeding;

NP 750 - Cattles feed with a standard diet with 1 x 10CFU Lactobacillus acidophilus strain NPC 750 mixed in water and added to the diet at the time of feeding.

The result of the study was explained as: "Just 14 d after initiating treatment, significant (P < .05) differences were observed among the three treatment groups. At this sampling time 56.6% of the control animals were positive, whereas only 20% of the animals fed with the NPC 747 sample and 11% of those fed with the NPC 750 probiotic were positive.
[0012] Comparing the data based on a positive pen basis, significant (P < .05) differences were also observed. Forty-one percent of the pens receiving the NPC 750 treatment had at least one positive animal, which was significantly (P < .05) the percentage of pens in cattle receiving NPC 747 (66% with at least one positive sample).'' Expressed in log units, the best data reduces the number of positives by around 0.5 logs (1 log is a ten times reduction).

At the filing date of the present invention the study was published on the Internet at the address: http://www.amif.org/ProbioticsReport042302.pdf.


[0013] Ongoing work has shown that levels of E. coli increase in cattle during the finishing period. Feeding of specific strains of beneficial bacteria has shown to reduce the levels of pathogenic proliferation. Studies of this nature are eliciting positive responses from a number of meat packers. Many packers are making strong recommendations to their supplying feedyards to feed probiotics to help with this issue. Their position is that if the level of E. coli entering the facility via the animal is reduced, their ability to further reduce contamination is vastly improved.
[0014] The research into this area is ongoing by universities and a number of companies. In particular several bacterial strains developed by Lallemand Animal Nutrition (LAN; Milwaukee, WI) have shown significant results in reducing the concentrations of E. coli O157:H7 and Salmonella via a process known as competitive exclusion. Competitive exclusion is a process by which beneficial bacteria are used to colonize the lining of the intestinal walls, reducing the area available for attachment by pathogenic microbes. The results so far confirm earlier theories that part of the effect noted through the feeding of beneficial bacteria results from this reduction in the area of the intestinal lining available to the pathogen for attachment.
[0015] Certain, specific strains of Lactobacilli and Propionibacterium developed by LAN have proven effective at reducing the numbers of these pathogens under different environmental conditions. Probiotic research has shown the effectiveness of gut colonization of beneficial bacteria in reducing pathogenic populations through competitive exclusion of these harmful organisms. In recent in vitro collaborative work by LAN and AgTech (Waukesha, WI., a 15 year-old biotech research company), it was found that several bacterial strains were highly effective in inhibiting the growth and development of strains of Salmonella and E. coli including E. coli O157:H7. The results indicate that in particular the BG2FO4 strain of Lactobacillus Acidophilus was very effective in inhibiting all strains of pathogenic E. coli tested. It is also important to note that the inhibition was a result, not only of competitive exclusion but also a result of the action of extracellular bacteriocins produced by the Lactobacillus. The results also indicated inhibition of several strains of Salmonella. A concluding result was that Lactobacillus Acidophilus BG2FO4 exhibits a high degree of pathogen oriented anti-microbial activity and is an excellent choice for use in beef cattle for this purpose.
[0016] The issued patent US5718894 describes a formulation for use in the promotion of growth or weight gain in a farm animal. The formulation comprises two groups of bacteria. A so-called first bacterium capable of producing lactic acid in the gastrointestinal tract of the animal and a second bacterium capable of producing a bactericide to which the bacteria are resistant, wherein said second bacterium is a Bacillus. The bactericide produced by the bacillus strain is capable of combating microorganisms that are the positive agent of enteric disorders, e.g. Staphylococcus aureus, E. coli and Salmonella (see column 2, lines 23-31). Examples of bacterium capable of producing lactic acid are bacteria of the genus Lactobacillus or Enterococcus. These are used to produce lactic acid and thus reduce the local pH in the gastrointestinal tract of the animal (see column 2, lines 8-22). A specific formulation for use in pigs is described. It is composed of the four strains Lactobacillus, Enterococcus faecalis, Enterococcus faecium and Bacillus licheniformis. Each strain is used at 10 cfu/g.
[0017] In summary current research has already and continues to reveal useful methodologies for the control of pathogenic bacterial populations in farm animals such as beef cattle. In relation to use of probiotic bacteria relevant detailed studies have mainly focused on use of suitable Lactobacillus Acidophilus strains.

SUMMARY OF THE INVENTION


[0018] The problem to be solved by the present invention may be seen in the provision of an farm animal feed composition that has an improved ability with respect to decreasing the number of Escherichia coli O157: H7 in the farm animal (preferably a cattle) when the farm animal were challenged with the Escherichia coli O157:H7 pathogen.


[0019] A solution to this is based on that the present inventors have identified that a feed composition comprising Enterococcus strains works better than a corresponding feed composition comprising similar amounts (CFU/g) of Lactobacillus Acidophilus strains.
[0020] Working examples herein demonstrate that cattle feed with around 10 cfu Enterococcus bacteria per day had a significant reduction of the number of Escherichia coli O157:H7 quantified in the faeces of the challenged animals. The reduction was around 1.5 to 2 logs in 10 to 14 days. 1 log unit denotes a ten times reduction and 2 log units denotes a 100 times reduction.
[0021] The above mentioned published corresponding example of an animal feed composition comprising Lactobacillus Acidophilus resulted in around 0.5 log reduction (see the Mindy Brashears et al. study discussion above). Without being limited to theory, it is believed that direct-feed microbial (DFM) products currently being commercially sold in cattle to reduce the number of Escherichia coli are based on Lactobacillus acidophilus, and they reduce the number of the target pathogen only by 0.5 logs at best.
[0022] In relation to the reduction of the number of Escherichia coli O157:H7, Enterococcus bacteria work better than Lactobacillus acidophilus bacteria. However, apart from Enterococcus the feed product may comprise smaller amounts of Lactobacillus acidophilus bacteria.
[0023] Accordingly, a first aspect of the invention relates to a farm animal feed product comprising at least 10 CFU/g feed of probiotic Enterococcus bacteria, characterized in that the product has at least 2.5 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed.
[0024] A second aspect of the invention relates to a method for feeding a farm animal comprising feeding the farm animal with a farm animal feed product comprising at least 10 CFU/g feed of probiotic Enterococcus bacteria, characterized in that the product has at least 2.5 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed.

DEFINITIONS


[0025] Prior to a discussion of the detailed embodiments of the invention is provided a definition of specific terms related to the main aspects of the invention.


[0026] The term "probiotic" is a well-defined term in the art and relates to a microorganism that confers health benefit to a farm animal when it has been in physical contact (e.g. when eaten) with the animal.
[0027] The term "Enterococcus" is a well-known and well-defined term for this Enterococcus genus of bacteria species. For further details see e.g. the standard reference book Bergeys Manual of Systematic Bacteriology. Based on his general knowledge, the skilled person is perfectly capable to determine whether or not a specific Enterococcus bacterium of interest is a bacterium of the Enterococcus genus.
[0028] The term "CFU" denotes Colony Forming Units.
[0029] Embodiments of the present invention are described below, by way of examples only.

DETAILED DESCRIPTION OF THE INVENTION

Farm animal feed product

[0030] The farm animal feed product should comprise suitable farm animal feedstuff ingredients. The skilled person is aware of selecting the adequate ones in relation to the specific farm animal of interest. Herein, such suitable farm animal feedstuff ingredients may be termed farm animal feedstuff ingredients known per se.


[0031] These ingredients should preferably be in concentrations adjusted to meet animal's dietary requirements and may include nutrient ingredients such as animal protein products, at about 0-95 weight percent; plant protein products, at about 0-95 weight percent; poultry egg products, at about 0-25 weight percent.
[0032] Further, the farm animal feed product may also comprise other suitable ingredients such as antibiotics such as Sarafin, Romet, Terramycin at about 0.01-50 weight percent; cyanocobalamin at about 40-60 mg/kg; D-biotin at about 5-20 mg/kg; D-pantothenic acid at about 250-350 mg/kg; folic acid at about 10-30 mg/kg; L-ascorbyl-2-polyphosphate (STAY-C, stable form of vitamin C) at about 1,000-4,000 mg/kg; myoinositol at about 3,000-4,000 mg/kg; niacin at about 600-800 mg/kg; p-amino-benzoic acid at about 350-450 mg/kg; pyridoxine hydrochloride at about 40-60 mg/kg; riboflavin at about 125-175 mg/kg; thiamine hydrochloride at about 50-80 mg/kg; choline chloride at about 6,500 7,500 mg/kg.

The farm animal feed product may be present in any suitable form, such as a powder, liquid or in form of pellets or tablets.


[0033] A preferred farm animal feed product is a composition comprising Enterococcus bacteria in a bolus, preferably a gelatin bolus. A most preferred farm animal feed product is a composition comprising Enterococcus bacteria, Glucidex IT12 (around 30%) and Type 4A Act Molecular Sieve Powder (around 10%).
[0034] The farm animal feed product may be in form of e.g. two different compositions one comprising the suitable farm animal feedstuff ingredients and the other comprising the Enterococcus bacteria as described herein. In such a case the farm animal feed product is comprised by such two compositions and accompanied by suitable instructions to administer them to the farm animal either simultaneously or sequentially. In other words, while the farm animals are fed with the suitable farm animal feedstuff ingredients they should also be fed with the probiotic Enterococcus bacteria as described herein.
[0035] Alternatively, the farm animal feed product may be in form of a composition comprising the suitable farm animal feedstuff ingredients and the probiotic Enterococcus bacteria as described herein. This may e.g. be in the form of a suitable powder, a liquid or in the form of pellets or tablets.
[0036] In order to e.g. improve some stability aspects of the probiotic bacteria it may be advantageous to provide the farm animal feed product as an stable emulsion of solids in-oil comprised by lipid soluble bioactive compounds such as inhibitory furanones dissolved in lipid forms of the continuous phase and with dry feed ingredients and the probiotic bacteria of interest forming the dispersed phase of the stable emulsion. See e.g. W002/00035 for further details.
[0037] Further, the farm animal feed product may be in form of a capsule e.g. a microencapsulated product.
[0038] As described above, a main point of the invention is to use Enterococcus bacteria instead of the Lactobacillus acidophilus bacteria as described in the prior art.
[0039] Accordingly, the farm animal feed product as described herein preferably comprises at least 5 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed, more preferably comprises at least 50 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed, even more preferably comprises at least 500 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed and most preferably comprises at least 5000 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed.
[0040] Said in another way, the most preferred farm animal feed product does not comprise significant amounts of Lactobacillus acidophilus. Such a most preferred farm animal feed product may be denoted a farm animal feed product consisting of at least 10 CFU/g feed of probiotic Enterococcus bacteria.
[0041] Preferably, the farm animal feed product comprises the probiotic Enterococcus bacteria in a concentration of at least 10 CFU/g feed, more preferably in a concentration of at least 10 CFU/g feed, even more preferably in a concentration of at least 10 CFU/g feed and most preferably in a concentration of at least 10 CFU/g feed. Generally, the farm animal feed product comprises the probiotic Enterococcus bacteria in a concentration of less than 10 CFU/g feed. In a preferred embodiment, the farm animal feed product comprises the probiotic Enterococcus bacteria in a concentration from 10 CFU/g feed to 10 CFU/g.

Probiotic Enterococcus bacteria


[0042] The probiotic Enterococcus bacteria may be any probiotic Enterococcus bacteria. Based on the herein disclosed information the skilled person is capable of selecting a specific Enterococcus strain of interest.


[0043] Preferably, the Enterococcus strain is an Enterococcus faecium.
[0044] Preferred Enterococcus faecium strains are Sf273 (CHCC 4202) and Sf301 (CHCC 3828). The farm animal feed product preferably comprises both Sf273 (CHCC 4202) and Sf-301 (CHCC 3828) in a ratio of preferably 50:50 (based on potency). The public deposition number of Sf301 (CHCC 3828) is DSM4789. The public deposition number of Sf273 (CHCC 4202) is ATCC27273.
[0045] Preferably, the Enterococcus bacteria are selected to be tolerant of the following conditions: high acid (pH 4.0), high concentrations of volatile fatty acids (200 to 400 mM mix-tures of acetic, propionic and butyric acids) and complete anaerobiosis.
[0046] Working example 2 herein, gives an example of a preferred assay to test if an Enterococcus bacteria is tolerant to these conditions.
[0047] More preferably the Enterococcus bacteria are also oxygen scavengers. Such bacteria are much more stable to oxygen exposure, moisture and heat than L. acidophilus.
[0048] Based on the information provided herein, combined with the common knowledge of the skilled person, it is routine work to select Enterococcus bacteria tolerant to the conditions given above.
[0049] Combined, these traits will confer a high degree of survival within the gastrointestinal tract of feedlot cattle. The product bacteria will be metabolically active upon ingestion by the animal. This activity can have an immediate impact either on the environment within the gastrointestinal tract, or on the E. coli cells present or on the receptor sites/niches with which the E. coli cells associate. Or, it could be any combination of these influences.

Farm animal


[0050] The farm animal may be a pig, a cow, a cattle, a sheep, a chicken, a duck, or an ostrich. More preferably the farm animal is a ruminant animal, in particular a cattle or a cow. Most preferably the farm animal is a cattle.

A method for feeding a farm animal comprising feeding the farm animal with a farm animal feed product as described herein

[0051] The feeding may be done according to the art and the skilled person is aware of how to properly feed farm animals.


[0052] Preferably, the farm animals are fed with an amount of farm animal feed product that provides at least 10 CFU Enterococcus bacteria per animal per day, more preferably the farm animals are feed with an amount of farm animal feed product that provides at least 10 CFU Enterococcus bacteria per animal per day, even more preferably the farm animals are feed with an amount of farm animal feed product that provides at least 10 CFU Enterococcus bacteria per animal per day and most preferably the farm animals are feed with an amount of farm animal feed product that provides at least 10 CFU Enterococcus bacteria per animal per day. Generally, the farm animals are fed with an amount of farm animal feed product that provides less than 10 CFU Enterococcus bacteria per animal per day. In a preferred embodiment, the farm animals are fed with an amount of farm animal feed product that provides from 10 CFU to 10 Enterococcus bacteria per animal per day, more preferably the farm animals are feed with an amount of farm animal feed product that provides from 10 CFU to 10 Enterococcus bacteria per animal per day.
[0053] The animals may preferably be fed once a day. Alternatively they may be fed twice a day or once every second day. The skilled person is aware of what is best in relation to a specific farm animal of interest.
[0054] Preferably, the farm animals are fed with the farm animal feed product as described herein at least once a day for at least 10 days, more preferably the farm animals are fed with the farm animal feed product as described herein at least once a day for at least 20 days.
[0055] Preferably, the farm animals are fed with the farm animal feed product as described herein until slaughter. In other words, the farm animals are preferably feed with the farm animal feed product as described herein at least once a day for at least the last 10 days until slaughter, more preferably the farm animals are feed with the farm animal feed product as described herein at least once a day for at least the last 20 days until slaughter.
[0056] Preferably, the farm animal feed product are used for feeding the animals in an amount and for a number of days where the animal feed reduces the number of Escherichia coli O157:H7 cells quantified in the faeces of the challenged animals by at least 1.5 logs, more preferably at least 2 logs.

Working examples herein describes a suitable assay to quantitatively measure this.

EXAMPLES

Example 1: Dose Titration Study to determine the effect of two different dosing regimens of a characterized Direct Fed Microbial Culture on the post-challenge fecal shedding of Escherichia coli O157: H7

Bacteria containing feed products:

[0057] CHB DFM: Two Enterococcus faecium strains present together at 50:50 (based on potency) in a gelatin bolus. The strains are Sf273 (CHCC 4202) and Sf-301 (CHCC 3828). The two Enterococcus faecium strains each are present in the product at 2.5 x 10 CFU/g.

CHB Probios TC: A corresponding product, which comprises probiotic Enterococcus faecium bacteria in a similar CFU/g as for CHB DFM. This product also comprised active dry yeast at around 2.5 x 10 CFU/g.

Objective:


[0058] The objective of this project was to explore the effect of two different doses of CHB DFM on the magnitude of fecal shedding of Nai E. coli O157: H7 in beef cattle fed a highly fermentable complete ration.

Materials and Methods:

[0059] Twelve beef steer calves weighing approximately 400 pounds were ranked by body weight and randomly allotted to one of three treatment groups. The treatment groups were control (no DFM) and DFM dosed at 2, or 20 g/head/day. All calves were orally dosed with DFM using gelatin boluses in order to assure that they received their daily target dose of DFM.

A similar strategy was used for Probios TC and Probios TC was dosed at 2, or 20 g/head/day.
[0060] Cattle were housed together in a single isolation room and allowed to commingle throughout the study. All cattle were allowed to consume a ration formulated with rolled corn and corn gluten feed containing sodium monensin (30 grams/ton). Fecal samples were collected and provided to detection of E. coli O157: H7 by enrichment technique to assure that calves were free of (Nal) E. coli O157: H7 prior to challenge.
[0061] Prior to the initiation of the study, all cattle were identified using duplicate unique Temple TM ear tags and were vaccinated with a modified live virus vaccine containing IBR, BVD, PI3, and BRSV (Bovishield 4 TM , Pfizer), 2 ml intranasal vaccination containing IBR and PI3 (TSV-2 TM , Pfizer), a single 2 ml injection of a Clostridial vaccine (Vision-7 TM , Intervet), and a single injection of anthelmintic to control internal and external parasites (Dectomax TM , Pfizer).

Experimental Design:


[0062] The following treatment groups were evaluated.

Treatment A: Non-medicated Control (n = 4)

Treatment B: CHB - Direct Fed Microbial (CHB-DFM) at a rate of 2 grams/head/day (n = 4)

Treatment C: CHB - Direct Fed Microbial (CHB-DFM) at a rate of 20 grams/head/day (n = 4)

Treatment D: Probios TC at a rate of 2 grams/head/day

Treatment E: Probios TC at a rate of 20 grams/head/day

[0063] Animals were inoculated with E. coli O157: H7, strains FRIK 1123 and FRIK 2000 which were adapted to nalidixic acid (Nal) in the laboratory (20 mu g/ml). The organisms were grown in GN broth (Difco laboratories, Detroit, MI.) for 7 h (approx. 0.8 abs at 600 nm), the two cultures were pooled and colony counts of the pooled cultures were done by spread plate technique. Each animal was inoculated (day 0) by using a stomach tube through a Frick speculum with 60 ml of the pooled cultures containing 8.6 x 10 CFU/ml of Nal E. coli O157:H7 (5.2 x 10 CFU/animal). Following administration of the challenge material, the tube was flushed with 120 mL of sterile phosphate buffered saline.


[0064] Animals were evaluated daily for evidence of adverse reactions. Fecal (rectal) specimens were collected on 1, 3, 5, 8, 10, 12, 14, 17, 19, 23, 24, 26, and 30 days following oral challenge. Fecal samples were placed in whirl paks, packed in ice and provided for detection and quantification of Nal E. coli O157: H7.

Detection and enumeration of Nal E. coli O157:H7:


[0065] One gram of feces was added to 9.0 ml of GN broth containing 50 mu l (0.05mg/liter) of cefixime (C), 200 mu l (10mg/liter) of cefsulodin (C), and 100 mu l (8mg/liter) of vancomycin (V). Samples were vortexed for 30 sec, serially diluted, and 100 mu l of 10, 10, 10 dilutions was spread plated, in triplicate, onto sorbitol MacConkey agar (SMAC) plates containing 20 mu g/ml of Nal. The remaining GN broth was incubated as an enrichment step in the isolation procedure. After 6 h incubation at 37 DEG C, 1.0 ml was transferred into 9.0 ml of GNccv broth and incubated an additional 18 to 24 h at 37 DEG C. The inoculated SMAC plates were incubated for 24 h at 37 DEG C and typical sorbitol-negative (gray colored) colonies were counted. A maximum of three colonies per sample per animal were collected, streaked onto blood agar plates, and incubated for 24 h at 37 DEG C. The indole test was done on colonies from the blood agar plates; indole positive colonies were tested for agglutination specific for O157 (Oxoid Diagnostic Reagents, Basingstock, Hampshire, England).

If E. coli O157:H7 colonies were not detected by direct plating (detection limit > 10/g), GNccv broth incubated for 18 to 24 h was plated, in duplicate, on SMAC plates containing Nal (20 mu g/ml) and incubated for 24 h at 37 DEG C. Following incubation, three colonies per sample with typical colony morphology (from the enriched samples) were streaked on blood agar plates and incubated for 24 h at 37 DEG C. The indole test was done on colonies from the blood agar plates and indole positive colonies were tested for agglutination specific for 0157.

Statistical Analysis:


[0066] The study had two outcomes of interest. First, the level of fecal E. coli O157:H7 shedding was compared between treatment groups. The independent variables were treatment, day, and the treatment by day interaction. The comparison was done using repeated measures analysis of variance (MIXED procedure, SAS Institute Inc.). The outcome was the level of fecal shedding (CFU/g of feces) with group as the treatment and day as the repeated measure. Day and treatment interaction was included as a fixed effect. Colony counts were log transformed prior to analysis.

Results:

[0067] Cattle in all groups shed at least 10 CFU/g (range 10 to 10) of Nal E. coli O157: H7 in the feces during the first week (days 1, 3 and 5) after inoculation. After that there was a general decrease in magnitude of shedding and numbers of Nal E. coli O157:H7 recovered ranged from 10 to undetectable (see Table 1). After day 12, the shedding pattern was somewhat erratic with concentrations fluctuating from 10 to undetectable. Treatment groups fed DFM at 2 or 20 g shed lower concentrations of Nal E. coli 0157: H7 in the feces compared to the control (P values = 0.01 and 0.06, respectively). The extent of reduction was greater in the group dosed with 20 g compared to 2 g dose. Table 3 reports data from a similar study made during another period than the study behind the data of Table 1. This second data confirms the overall results described above.

Conclusions:

[0068] DFM at 20 g per animal per day caused a significant reduction in the level of shedding of Nal E. coli O157:H7.

Id=Table 1. Columns=5

Effects of Direct-fed microbials (DFM) on fecal shedding of Nalidixic acid-resistant E. coli O157:H7 in cattle.

Head Col 1: Sampling days

Head Col 2 to 4: DFM, g/animal/day

SubHead Col 1:

SubHead Col 2: 0

SubHead Col 3: 2

SubHead Col 4: 20

03.5 x103.5 x103.5 x10

12.5 x10 a1.5 x10 a3.5 x10 a

31.5 x10 a9.1 x10 a2.3 x10 a

56.5 x10 a1.6 x10 a2.2 x10 a

87.6 x10 a6.0 x10 a3.0 x10 a

103.9 x 10 a, b5.7 x10 a5.4 b

123.3 x10 a3.0 x10 a0 b

141.8 a2.9 a9.4 x10 a

177.4 x10 a9.3 a5.3 x10 a

191.9 x10 a2.6 x10 a2.9 b

234.5 x10 a1.2 x10 a1.1 x10 a

245.4 a2.0 a1.6 x10 a, b

266.3 x10 a9.5 a1.5 a

300 a0 a1.8 a


Treatment effect P= 0.02

Days effect P < 0.01

Treatment by Days Interaction P = 0.6

a, b Means not sharing the same letters differ at P < 0.1.

Id=Table 1. Columns=3

Treatment Means

Head Col 1: Treatment

Head Col 2: Mean (CFU/g of feces)

Head Col 3: Significance

Control, 0 g/day1.9 x10Control vs. 2 g DFM P = 0.06 Control vs. 20 g DFM P = 0.01

DFM, 2 g/day4.5 x102 g DFM vs. 20 g DFM P = 0.54

DFM, 20 g/day2.8 x1020 g DFM vs. 200 g DFM P = 0.01


P-value (treatment effect) = 0.02.

Id=Table 3. Columns=6

Effects of Direct-fed microbials (DFM) on fecal shedding of Nalidixic acid-resistant E. coli O157:H7 in cattle.

Head Col 1: Day

Head Col 2: Control

Head Col 3: DFM 2gX

Head Col 4: DFM 20g

Head Col 5: TC 2g

Head Col 6: TC 20g

D037000003700000370000037000003700000

D1340000012000024000007500003700000

D3900000120000580000100000580000

D58300033000250000600003800000

D850004701400016035000

D102800120721300520

D12140011010011000570

D1520001307120620

D1912035019170017

D22171721189

D2423023001814

D26961000

Example 2: Assay to select Enterococcus bacteria that are tolerant to preferred conditions:


[0069] A number of publicly avialable Enterococcus bacteria were tested in the assay described below. The strains SF273 (CHCC4202) and SF301(CHCC 3828) were both tolerant to the below described testing conditions.

Exposure to Rumen Fluid

[0070] The medium of Bryant MP and Burkey LA (J. Dairy Science 1960) containing 40% rumen fluid (RF medium) was prepared either under 100% CO2 (with sodium carbonate solution as buffer) or under 80%:20% N2:CO2 headspace gas (with sodium bicarbonate solution as buffer). This medium was used to score growth of the test strains as 0 (no growth) or +, ++, +++, or ++++ (excellent growth) at 37C after 48 to 72 h.

Exposure to Volatile Fatty Acids

[0071] Medium 10 of Bryant MP and Robinson IM (J. Dairy Science 1966) was prepared to test the tolerance of each test strain to volatile fatty acids (acetic, propionic and butyric acids, 200 to 400 mM). This medium was used to score growth of the test strains as 0 (no growth) or +, ++, +++, or ++++ (excellent growth) at 37C after 48 to 72 h. Only those strains having a +++ or ++++ score advanced in the testing procedure are in present context an Enterococcus bacterium considered tolerant high concentrations of volatile fatty acids (200 to 400 mM mix-tures of acetic, propionic and butyric acids).


[0072] Acid Tolerance
[0073] Medium 10 Bryant MP and Robinson IM (J. Dairy Science 1966) was prepared and poised at pH 4.0, 5.0, or 6.0 using a 5 N solution of HCl (hydrochloric acid). The medium was autoclaved, cooled and then inoculated with each test strain. These media were used to score growth of the test strains as 0 (no growth) or +, ++, +++, or ++++ (excellent growth) at 37C after 48 to 72 h. Only those strains having a +++ or ++++ score in the pH 4.0 medium advanced in the testing procedure are in present context an Enterococcus bacterium considered tolerant of high acid (pH 4.0) concentrations.

Oxygen Tolerance


[0074] Medium 10 of Bryant MP and Robinson IM (J. Dairy Science 1966) was prepared to test the tolerance of each test strain to oxygen tolerance. In this test, the medium was modified as follows: 2% w/v agar was added to solidify the medium, the amount of resazurin (redox indicator) was doubled, and the amount of reductant, cysteine hydrochloride solution, was reduced by 50%. This medium was dispensed in 10 mL amounts and solidified after autoclaving in an upright position. Each test strain was grown in broth culture overnight and then stab-inoculated from top to bottom in the center of the 10 mL tubes of modified Medium 10. The inoculated tubes were exposed to the atmosphere for 5 min then closed and incubated overnight (ca. 18 h) at 37C. After incubation the top one-centimeter of the medium had oxidized (turned the redox indicator pink) and the resultant growth pattern of each test strain was scored. The strain was scored as a strict anaerobe if visible growth along the stab line was only in the reduced portion of the tube. The strain was scored as an oxygen tolerant anaerobe if strain growth extended into the pink (oxidized) portion of the medium. The strain was scored as a facultative anaerobe if growth was visible along the stab line within both the reduced and oxidized portions of the tube. The strain was scored as an aerobe if growth was visible only within the oxidized portion of the medium. In the present context an Enterococcus bacterium is considered tolerant to complete anaerobiosis conditions if it grew in the reduced portion of the medium.

Oxygen scavenging

[0075] Medium 10 as modified for oxygen tolerance testing was used. After oxygen tolerance capacity was scored, the stab-inoculated tubes were re-incubated at 37 C for an additional 24 h. Then the oxidized zone of each tube was examined and scored as follows. If the pink zone was re-reduced to colorless, then the strain was scored as being an oxygen scavenger (characteristic present or absent). In the present context an Enterococcus bacterium is considered an oxygen scavenger if it re-reduced the medium.

REFERENCES

[0076] The American Meat Institute (AMI) Foundation published 2002 result of a research study that was done by Mindy Brashears and Michael Galyean of Texas Tech University. At the filing date of the present invention the study was published on the Internet at the address: http://www.amif.org/ProbioticsReport042302.pdf.Claims:

1. A farm animal feed product comprising at least 10 CFU/g feed of probiotic Enterococcus bacteria, characterized in that the product has at least 2.5 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed.
2. The farm animal feed product of claim 1, wherein the product comprises farm animal feedstuff ingredients known per se.
3. The farm animal feed product of claims 1 or 2, wherein the product has at least 5000 times more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as CFU/g feed.
4. The farm animal feed product of any of the preceding claims, wherein the probiotic Enterococcus bacteria is Enterococcus faecium bacteria.
5. The farm animal feed product of any of the preceding claims, wherein the Enterococcus bacteria are tolerant to the following conditions: high acid (pH 4.0), high concentrations of volatile fatty acids (200 to 400 mM mix-tures of acetic, propionic and butyric acids) and complete anaerobiosis.
6. A method for feeding a farm animal comprising feeding the farm animal with a farm animal feed product according to any of the claims 1-5.
7. The method for feeding a farm animal of claim 6, wherein the farm animal is a cattle.
8. The method for feeding a farm animal of claims 6 or 7, wherein the farm animals are feed with an amount of farm animal feed product that provides from 10 CFU to 10 Enterococcus bacteria per animal per day.
9. The method for feeding a farm animal of any of claims 6-8, wherein the farm animals are feed with the farm animal feed product at least once a day for at least 10 days, more preferably the farm animals are feed with the farm animal feed product at least once a day for at least 20 days.
10. The method for feeding a farm animal of any of claims 6-9, wherein the farm animal feed product are used for feeding the animals in an amount and for a number of days where the animal feed reduces the number of Escherichia coli O157:H7 cells quantified in the faeces of the challenged animals by at least 1.5 logs, more preferably at least 2 logs.

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