Note: Descriptions are shown in the official language in which they were submitted.
WO 92/12639 PCT/l.'S92/00708
_1_
FEED ADDITIVE AND METHOD
FIELD OF INVENTION
This invention relates to a new method for delivering
viable microbial cells in animals' diets and for reducing
microbial pathogens such as Salmonella.
BACKGROUND INFORMATION
Under certain conditions some members of the
indigenous gastrointestinal microbiota can become
opportunistic pathogens causing, a variety of enteric
diseases. More often, however, pathogens gain access to
the GI tract as contaminants in food or water. Notable
among the latter are a number of bacterial genera including
Escherichia, Salmonella, Shigella, Yersina, yibrio,
Camgylobacter and Clostridium, as well as viruses (e. g.,
1S roto-, astro- and ciliciviruses) and intestinal parasites
(e. g., Giardia and Entamoeba species). Acute and chronic
enteric diseases caused by these and other microorganisms
occur worldwide causing considerable human misery and loss
of economically important animals. Certain microbial
activities have also been associated with production of
mutagens within the GI tract.
It is known that other members of the indigenous
microbiota exist in a symbiotic or synergistic relationship
with their host contributing in many positive ways to the
host's general health and well-being. It is well-known
that germ-free animals are very susceptible to pathogens
and have poorly developed GI tracts. In return for the
nutrient-rich and stable eeosystem provided for them, the
indigenous microbiota can provide their hosts with an
assortment of benefits including among others protection
against enteric pathogens to process known as colonization
resistance or competitive exclusion), stimulation of normal
. development and function of the GI mucosa, production of
various vitamins and other nutrients, and re-metabolism of
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the host's abundant endogenous mucosal tissue.
It has been reported on numerous occasions that the
enteric lactobacilli (i.e., bacteria belonging to the genus .
Lactobacillus which reside in the GI tract and which
include a large number of nonpathogenic, non-toxic
bacteria) play an important role in the health and well-
being of their human and animal hosts.
The metabolic endproducts of Lactobacillus metabolism,
such as acetic acid, lactic acid and hydrogen peroxide, are
well-known for their antimicrobial activities. They are
believed to play a significant role in maintaining proper
conditions within the GI tract. Some lactobacilli produce
bacteriocins or bacteriocin-like proteins which also
exhibit bacteriocidal activity toward other members of that
species or closely related species. Reports have appeared
concerning low molecular weight, antimicrobial substances
produced by lactobacilli. With the exception of reuterin
which is produced by Lactobacillus reuteri, none of these
low molecular weight substances has been identified and
these reports have not been confirmed. In fact, some of
these substances have proven to be none other than lactic
acid, acetic acid or hydrogen peroxide.
Some of these beneficial microorganisms have been used
as probiotics. The term "probiotics" is attributed to
Parker (32) who defined probiotics as "organisms and
substances which contribute to intestinal balance" when
used as dietary supplements. This publication and all
other publications and patents cited herein are
incorporated' herein by reference. Later, Fuller (17)
considered this definition to be too broad since, in .
addition to including cell cultures and microbial
metabolites, it could encompass antibiotic preparations.
More recently, a number of summaries have appeared in the
literature describing the scientific basis for use of
probiotics as intestinal inoculants for production animals
( 15, 40 ) . It has been suggested that the term "probiotics"
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«'O 92/12639 PCTlL'S92/00708
be replaced by the term °'direct feed microorganisms," or
DFM's (14).
It is generally held that during periods of low
resistance, such as stress, undesirable microorganisms are
able to proliferate in the GI tract of animals, humans
included. Maintaining a normal, healthy balance of
microorganisms is deemed to be critical during such
stressful periods (15). The concept underlying use of
DFM's, therefore is that if sufficient numbers of an
appropriate microorganisms) are introduced into the
intestinal tract (i) at times of stress and/or disease,
( ii ) at birth, or ( iii ) after antibiotic treatment ( when
minimal LAB are present), the negative consequences of the
microbial imbalances can be minimized or overcome. Using
such preparations of live, naturally occurring
microorganisms helps restore and maintain the proper
balance of beneficial microbes in the GI tract during times
of stress, disease, and following antibiotic therapy (15).
This concept, descriptions of proposed modes of action, and
evidence for the efficacious uses of DFM~s for all
production animals are summarized in reviews by Fox (15),
Sissons (40); and by various authors (36).
The concept of adding viable, harmless lactic acid
bacteri a to the gastrointestinal tract as a dietary
supplement was first appreciated by Metchnikoff (26) who
viewed the consumption of yoghurt by Bulgarian peasants as
conferring a long span of life. Some workers have claimed
that the therapeutic value derived from ingestion of such
fermented milk products 3,s related to the viable bacteria
present in these products (18, 42). Since Metchnikoff~s
early reports, several studies have shown the ability of
lactobacilli, for example, to suppress coliform growth.
Feeding viable Lactobacillus acidophilus cells to young
dairy calves was shown to reduce the incidence of diarrhoea
( 4 ) , and increase the numbers of lactobacilli and reduce
coliform counts in feces ( 5 ) . These findings contrast with
WO 92/1?639 PCT/~'S92/00708
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' those of others who have been unable to demonstrate
,
benefits from feeding either Lactobacillus acidophilus (13,
21) or milk cultured with Lactobacillus acidophilus or
Lactobacillus lactis (27).
In a detailed study by Muralidhara et.al. (28),
piglets given a Lactobacillus lactis concentrate for up to
8 weeks after birth showed a progressive decline in
coliform counts in fecal samples. Scouring in these
animals was negligible, but was evident in control pigs
especially at weaning. Underdahl et al. (49) observed only
mild diarrhoea lasting 2-4 days in gnotobiotic pigs
inoculated with Streptococcus faecium prior to artificial
Escherichia coli infection. In the same study, persistent
diarrhoea occurred in pigs similarly infected with
Escherichia coli, but without prophylactic treatment with
the Streptococcus microorganism.
The lactic acid bacteria (LAB), particularly those
classified in the following genera, are often used in
probiotics: Lactobacillus, Lactococcus, and Enterococcus.
Included among these are the following species:
Lactobacillus acidophilus, Lactobacillus bulgaricus,
Lactobacillus plantarum, Lactobacillus casei, Lactobacillus
lactis, Lactococcus lactis, Lactococcus thermophilus,
Lactococcus diacetylactis, and Enterococcus faecium.
Besides these LAB, some species of Bacillus (Bacillus
subtilis, Bacillus t-oyoi) and yeasts and molds
(Saccharomyces cerevisiae, Aspergillus orryzae, and
Torulopsis sp.) are used as DFM's (15).
' ~ Certain Lactobacillus species in fact are added to
human and animal foodstuffs either to preserve them,
enhance their flavors and/or exert other beneficial effects
in the GI tract. Lactobacillus plantarum strains, for
example, are grown commercially in large amounts and used .
as starter cultures for the commercial preservation of a
variety of human foods (meats, vegetables, and dairy
products) and animal foods (silage).
~, ':: .' .: :~. . . " , ,, ..;.,
WO 92/12639 PC'~'/US92/00708
Lactobacillus acidophilus strains are grown
commercially in large amounts to be added to human (e. g.,
milk} or animal (feedstuffs) foods as a means of
introducing these bacteria into the GI tract where they can
exert beneficial effects. Although these bacteria are
likely to be already present in the GI tract their numbers
may vary widely. from individual to individual, and
therefore beneficial effects of these bacteria may not be
present in persons deficient in these bacteria. Reports on
the beneficial effects resulting from the oral
administration of live Lactobacillus cultures have
inereased in recent years with findings that dietary
Lactobacillus therapy affords protection from colon cancer
for human populations on western diets, reduces the
incidence of experimentally induced large bowel tumors in
rats, reduces the fecal concentration of bacterial enzymes
known to catalyze the conversion of procarcinogens to
proximal carcinogens in humans, and reduces the serum
cholesterol levels in swine.
Several studies have been conducted to determine the
effect of lactobacilli on the performance of domestic avian
species. Some of these studies indicate that dosing
broilers with L. acido hp ilus improved their growth (11,
48). An increase in egg production as a result of addition
of lactobacilli to laying hens feed has also been reported
(20).
Improvement of young turkeys body weight and feed
efficiency was obtained with a Lactobacillus product added
to the feed (16). Similar effects were reported (34) when
L. acidophilus in combination with varying protein levels
.was fed to turkeys up to 12 weeks of age, but no difference
with the controls was observed at 16 weeks. Dosage of 10'
colony forming units (CFU) and higher depressed chick
growth (51, 52}.
Other strains of lactobacilli did not stimulate
broilers weight gain (52), and did not stimulate egg
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production (19). Damron et al. (9) did not find any
beneficial effect of L. acidophilus and other lactobacilli
cultures in turkey breeder hens.
Nurmi and Rantala (29) demonstrated that the
intestinal microflora present in some adult chickens (i.e.
the cecal microflora) interferes with colonization by
salmonellae of newly hatched chicks. The application of
this concept, known as the Nurmi concept or competitive
exclusion, has been successfully tested in some
laboratories and is also used commercially (22, 41, 53,
54). There are many problems associated with this method,
in particular, a lack of adequate selective isolation and
characterization techniques to study and consistently
obtain cecal flora preparations (3, 25, 42).
Mannose and lactose were shown to significantly reduce
Salmonella typhimurium adherence to the ceca of chicks
(31). The inhibitory effect of these sugars was believed
to take place by blocking the receptor sites on the gut
epithelium and on the microorganism pili. It has been
shown that providing dietary lactose together with cecal
flora contents to broiler chickens reduced the occurrence
of Salmonella (7, 8, 31).
Historically, Lactobacillus administration (i.e.,
inclusion of viable cells in the feed) to animals has not
yielded consistent benefits. There are many reasons for
this including, for example, using Lactobacillus species or
strains unadapted to or unsuitable for the animal being
treated, or using conditions which do not produce a
colonization of the Lactobacillus within the GI tract.
One of the major problems or limitations encountered
in commercial scale application of DFM's to animals is (i)
the availability of suitable delivery systems , and ( ii ) the
ability to get the probiotic preparations to the animals as
quickly as possible after birth. This is particularly true
when pelletized feeds are used, as is the case in the
poultry industry. The pelletization process generally
CA 02100774 2001-09-17
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includes one or more heating steps involving temperatures
high enough to pasteurize or sterilize the feed components,
thereby precluding incorporation of viable microorganisms
into these feeds prior to pelletization.
. 5 The present invention describes novel methods and
processes for overcoming some of these problems, by
delivering viable DFM's in feed additives. Lactobacillus
reuteri, along with L. animalis, and L. salivarius, which
may be used in the invention, are naturally occurring
microorganisms in the GI tract of animals including
domestic avian species ( 38 ) . The DFM used to develop these
methods using pellets is Lactobacillus reuteri. This
species was chosen because it has demonstrated efficacy as
a DFM in poultry (33). This efficacy is also discussed in
PCT/US88/01423, filed April 28, 1988 and published November
3 . 1988 .
Lactobacillus reuteri is a species of lactic acid
bacteria recognized since the turn of the century (30).
Originally assigned different species names (e. g.,
Lactobacillus fermentum biotype II), it obtained distinct
species status in 1980 and is registered in the 1988
edition of Hergey's manual (23, 24). It is found in foods,
particularly dairy products and meats, but exists primarily
in the GI tract of healthy animals, including humans (1,
10, 12, 23, 24, 37, 38, 39, 50).
Lactobacillus reuteri is the dominant
heterofermentative Lactobacillus inhabiting the GI tract
(37, 38, 39). Lactobacillus reuteri is a symbiotic
resident of the gastrointestinal (GI) tracts of humans,
swine and other animals. The neotype strain of L. reuteri
is DSM 20016 (ATCC No. 53609). This strain and strain 1063
(ATCC No. 53608), discussed in the co-pending application,
are available to the public at the American Type Culture
Collection (Rockville, MD) having been deposited therein
April 17, 1987.
1V0 92/12639 fCT/(.'S92/li070H
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N
Lactobacillus reut~ri is a typical heterofermenter,
converting sugars into acetic acid, ethanol, and COZ in
addition to lactic acid which is the major endproduct of
homofermentative metabolism carried out by species such as
Lactobacillus acidophilus (21). It utilizes the
phosphoketolase pathway for conversion of glucose to
endproducts. When glycerol, an alternate hydrogen
acceptor, is present in the culture medium together with
glucose or other utilizable carbon and energy sources
(e. g., lactose), acetate rather than ethanol accumulates,
and the glycerol is reduced to 1,3-propanediol via the
metabolic intermediate, 3-hydroxypropionaldehyde (3-HPA).
3-HPA has been shown to have potent antimicrobial activity,
and Lactobacillus reuteri appears to be unique among
microorganisms examined to date in its ability to secrete
this substance, termed reuterin, into the surrounding
medium (2, 6, 12, 44, 45, 46, 47). This unique
antimicrobial activity may play a role in competitive
survival of this species in the gastrointestinal ecosystem,
and/or its ability to regulate growth and activities of
other microorganisms in this ecosystem (12). It is thus
very important to establish this microorganism early in
animals. It is therefore an object of the invention to
provide a method for delivering DFM's, such as
Lactobacillus, to avian species.
It is another object of this invention to provide a
food or feed additive formulation and method comprising
isolated and identified pure cultures of Lactobacillus
reuteri and/or other Lactobacillus species together with a
sugar source such as lactose, using whey as a source for
this sugar.
It is a further object of the invention to provide a
formulation that results in rapid weight gain for growing
animals.
It is a further object of the invention to provide a
formulation that decreases the number of pathogenic
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_g_
microorganisms in the gastrointestinal tract, with the
purpose of adding any sugar for at least the purpose of
being a source of carbohydrate for the metabolism of the
Lactobacillus but not utilized by the animal or the
unwanted microorganism(s).
"_ Other objects and advantages will be more fully
apparent from the following disclosure and appended claims.
SUMMARY OF INVENTION
The invention includes a formulated product that may
be used as an animal feed additive and that includes
Isolated and identified pure cultures) of naturally
occurring gastrointestinal microorganisms, for example,
Lactobacillus reuteri, L. animalis, and/or L. salivarius,
and a sugar source. The invention also includes a method
of feeding the formulation to animals. Preferably the
sugar source is whey, when animals which do not metabolize
lactose such as chickens are used, because whey contains
the sugar lactose and is an easily obtainable and
voluminous waste product.
A dietary supplement is prepared containing viable
cells of a DFM such as Lactobacillus reuteri, an oil and a
cryoprotectant such as whey powder. The Lactobacillus
cells may be coated on the surface of whey pellets or be
contained in the pellets. As used herein, the word
"pellet" means a compacted whey particle which may be of
any size or shape that is ingestible by the animal to be
fed the supplement.
The formulation of the invention when fed to animals
provides a means to decrease populations of undesirable
gastrointestinal microbes and results in increased weight
gain of the animals, especially under the less than optimum
growth conditions normally present in commercial livestock
environments.
_.-. CA 02100774 2001-09-17
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9a
This invention provides a method of establishing a
direct feed microorganism in the gastrointestinal tract of
an animal comprising adding lyophilized direct fed
microorganisms to a whey pellet, wherein the microorganism
is Lactobacillus reuteri.
This invention provides a feed additive,
comprising a pellet comprising a pure culture of
Lactobacillus reuteri and whey.
This invention provides a formulation for oral
administration to poultry, comprising: (a) a bacterial
culture comprising at least one live pure culture of a
Lactobacillus species which occurs naturally in
gastrointestinal tracts of poultry, said Lactobacillus
species selected from the group consisting of L. reuteri, L.
salivarius, and L. animalis; and (b) a source of sugar
metabolizable by the Lactobacillus species in the bacterial
culture but not metabolizable by poultry, wherein said
formulation is capable of reducing Salmonella in poultry and
provides a concentration of about 2% to about 5% of said
sugar in poultry feed when the formulation has been added to
said feed to provide a level of Lactobacillus sufficient to
reduce Salmonella in the gastrointestinal tract of poultry,
and wherein said formulation when fed to poultry results in
greater numbers of Lactobacillus cells in the
gastrointestinal tract than when poultry is fed
Lactobacillus cells without the sugar.
This invention provides a method of decreasing
numbers of undesirable microbes in an animal's
gastrointestinal tract, comprising: (a) obtaining at least
one pure culture of Lactobacillus reuteri cells; (b)
obtaining a source of sugar; and (c) administering the
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9b
Lactobacillus reuteri cells and the sugar orally to the
animal.
This invention provides a method of decreasing
numbers of undesirable microbes in gastrointestinal tracts
of animals, comprising: (a) obtaining at least one pure
culture of Lactobacillus reuteri cells; (b) obtaining a
source of sugar metabolizable by the Lactobacillus reuteri
cells and not to a significant extent by the animals or the
undesirable microbes; and (c) administering the
Lactobacillus reuteri cells and the sugar orally to the
animal.
This invention also provides for the use of the
aforementioned additives .
Other aspects and features of the invention will
be more fully apparent from the following disclosure and
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' ~ v : ~~=w appended claims .
DETAILED DESC~tIPTION OF THE INVENTION AND PREFERRED
EMBODIMENTS TFB;REOF
The present invention provides a formulation usable as
a food or feed additive for animals. In the broadest
aspect of the invention, animals may be fPd the additive in
a variety of ways: for example, (1) the additive may be
combined with dry feed during feed milling or when the feed
is' delivered to the animals; (2) the additive may be
sprinkled on the food as a powder; or (3) the additive may
be mixed in the drinking water. Preferably, to minimize
labor, the additive is mixed with dry feed.
In the particular invention, such animals specifically
include all poultry and mammals, including human beings.
In its most basic form, the formulation for a particular
animal comprises one or more pure cultures of a
Lactobacillus species naturally occurring in the
gastrointestinal tract of that animal and a source of a
sugar that is metabolizable by the Lactobacillus species
but not to any great extent by the animal. Thus, the
formulation of the invention comprises:
(a) a bacterial culture comprising at least one live
pure culture of a Lactobacillus species which
occurs naturally in a particular animal group;
and
(b) a source of sugar metabolizable by the
Lactobacillus species in the bacterial culture
but not metabolizable by the animals in the
group.
By the term "group" is meant animals of a particular
species or group of species which share in common a
tendency to have a similar gastrointestinal Lactobacillus
flora and a similar inability to metabolize a sugar which
is metabolizable by the Lactobacillus flora. As discussed
below, the formulation discussed in detail herein has been
. :; .
WO 92/12639 PCT/~,'S92/0070~
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devised for poultry but is adaptable to other animals, and
includes a source of lactose which is not metabolizable by
poultry.
In the preferred embodiment, the formulation comprises
a live, pure culture of at least one of Lactobacillus
reuteri, L. animalis and L. salivarius, and a sugar source.
The preferred sugar source is whey, because it is
inexpensive and easily available, and because it contains
lactose, a good source of carbon and energy for growth of
the added microorganisms. An additional advantage of using
a lactose source for feeding poultry or other birds is that
birds do not utilize this sugar, and it is therefore
readily available for the added microorganisms.
Preferably, powdered whey is utilized as the lactose source
to minimize shipping eosts and spoilage prior to
formulation of the additive.
The preferred method of formulating the additive is as
follows. L.reuteri, L.animalis and/or L.salivarius are
grown individually in a variety of appropriate media used
for lactobacilli. Lactose or maltose are the preferred
sources of energy so that the cells are capable of rapid
metabolism of the carbohydrates which may be present in the
formulation or in~the animals food. The cells used for
the preparation of the additive may be freshly harvested,
frozen, lyophilized or suspended in oil or a specifically
formulated diluent sueh as an aqueous solution.
Commercially available whey powder or whey concentrate is
used to formulate the additive. Although the cells and
whey may be fed separately, the are preferably mixed
together with or without other ingredients (e.g. corn, .
- soybean meal, wheat, etc.). 'The mixture may be of a
variety of microbe and whey mixtures, for example a solid
and a solid (e. g. fine powder with granulated whey, etc.),
a liquid and a solid ( cell suspension and whey ) , a solid
and a liquid (lyophilized cells and a liquid whey
concentrate) or a liquid and a liquid (liquid cell
WO 92/12639 PCT/~;S92/00708
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suspension and a liquid whey concentrate). The additive
final presentation of the mixture could be as a powder,
granules, or pellets or liquid. '
In its most preferable form, the invention comprises:
a method of delivering DFM's to birds so that the DFM's are
established in the gastrointestinal tract. L. reuteri
cells or other DFM's are incorporated onto the surface or
within pellets. The pellets may be fed to the birds, for
example poultry, along with the birds' normal diet.
Lyophilized (freeze-dried) Lactobacilli reuteri
strains, T-1 (isolated from turkey) and 11284 (isolated
from chickens), when held at room temperature
(approximately 25°C) are found to remain viable for as long
as 30 days but to decrease in number. For example, a
population of 6 x 106 colony forming units (GFU)/g were
recovered of the original 3 x 10'° CFU/g at 30 days. It was
found that when the lyophilized cells were suspended in an
oil, such as sunflower oil at room temperature for 30 days,
no loss of viability was observed.
The invention provides in its one preferred embodiment
that lyophilized L. reuteri cells suspended in oil are
coated over palletized whey particles. Under room
temperature, no decrease in viability is observed for up to
seven days. When the Lactobacillus coated palletized
particles of whey are mixed with poultry feed, no
significant loss of viability occurs over four days at room
temperature.
In another preferred embodiment of the invention
Lactobacillus reuteri cells in oil are mixed with whey
powder and then the mixture is compressed into pellets or
tablets. Although survival is lower than in the first
embodiment when there is no cooling in the pelletization
process, survival is sufficient for use of the pellets as
a beneficial food additive which aids in establishing the
DFM in the animal.
The features and advantages of the present invention
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will be more clearly understood' by reference to the
following examples, which are not to be construed as
limiting the invention.
Example 1. Growth of Turkey Poults to be Fed Additive
One day old Nicholas turkey tom poults are used in
this study. The poults are not toe clipped, desnooded or
wing clipped, nor are they given any vaccinations.
The turkeys are placed in animal rooms at the
Dearstyne Poultry Research Center, Department of Poultry
Science at NCSU's Agricultural Research Service (NCARS).
The animal rooms have controlled ambient temperature, day
length and thermostatically controlled Petersime brooding
batteries (Petersime Incubator Co., Petersime, OH).
A normal turkey starter diet, for example as shown in
Table 1 with and without whey powder, is used throughout
the trial: The amount of whey in the diet allows for a
final 2.2~ lactose. The trial is twenty days in duration,
covering the period from day of,hatch to day 12. The
turkeys are weighed on Day 0 (at hatch), Day 5, Day 12, Day
15 and Day 20.
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. .,~ .,' , . ,
TURKEY STARTER DIET .
INGREDTENT CONTROL PLUS WHEY
(lbs/1000) (lbs/1000)
Soy 490 487
Corn 405 373
Whey (73~ lactose) --- 30
IO Poultry Fat 44.4 49.6
Ethoxyquin 0.12 0.12
Dicalcium phosphate 35.5 35.5
Limestone 14.5 14.5
Sodium Chloride 3.7 3.7
Trace minerals 1.0 1.0
Vitamin mix, 1.0 I.0
Choline Cl (60~) 2.0 2.0
L-Lys.HCl 0.1 0.1
DL-Met 2.8 2.8
1000.0 1000.0
Example 2: Growth and Quantitation of Bacterial Cultures
L.reuteri 11284, known to colonize the chicken GI
tract, and L.reuteri Tl, which is a strain isolated from
turkeys, are the strains which are used. The
Lactobacillus strains are grown in LCM medium utilizing
lactose or maltose for 24 h at 37°C, harvested by
centrifugation, and washed twice with fresh basal medium as
previously described (2, 6). These cells are mixed into _
the animal feed at a level of approximately 105 CFU g-~ of
feed. This inoculum level has been shown to effectively ,
enhance the population level of this microorganism in the
chicken ceca (Casas et al., 1990, in preparation). The
number of L. reuteri in the feed and in the ceca are
~~0 9?/t2G39 PCT/L~S92/()0708
monitored as previously described (6). Appropriate
dilutions are plated onto LBS agar and Mincubated
anaerobically (Gas-Pak jars) at 37°C for 48 h. Plates
containing about 50 to 200 CFU are overlaid with glycerol
5 agar seeded with L. plantarum indicator cells, reincubated
anaerobically for 24 h, and colonies showing growth
inhibition zones counted as reuterin-producing L.reuteri
cells.
S. senftenberg, isolated from turkeys, resistant to
10 novobiocin and nalidixic acid, was obtained from Evillmar
Poultry Co. (Evillmar, MN). Inocula far infectious
challenge are prepared from cultures grown in BHI Broth
(Difco Laboratories, Inc., Detroit, MI) and incubated at
37°C for 24 hours. The cultures are diluted appropriately
15 in sterile 50 mM phosphate buffer, pH 7.0, to obtain
challenge inocula containing 106 CFU per ml. Enumeration
of these Salmonella is carried out by plating appropriate
dilutions on Salmonella medium (35).
Caecal content samples for microbiological enumeration
are prepared from sacrificed birds. Caeca are carefully
removed from the birds and the open end of each is clipped.
The exterior of the caecum is alcohol sterilized before
transferring its contents to a stomacher bag for mixing and
further dilution.
Example 3. Treatment of Poults
Turkey points of Example 1 are subjected to the
following eight treatments with two pens of 15 birds per
pen being in each group:
Salmonella senftenberg infected group
1. Control, no whey, no L.reuteri
2. No whey, L.reuteri
3. Whey, no L.reuteri
4. whey, L.reuteri
L.reuteri, when administered is mixed into the feed.
The inoc~slated feed is changed every two days to guarantee
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the presence of viable L. reuteri in the feed . Whey is
added to the feed, before milling, for a final 5% lactose
concentration. Alternatively, L. reuteri and concentrated
or dehydrated whey are formed into tablets , or added
together in any product in which both components in a
liquid or solid form have been previously combined, and the
combination added to the feed of the animals.
S. senftenberg (106 CFU per ml) is crop fed by the
means of an animal feeding stainless steel needle attached
to a hypodermic syringe on day 5 after hatch.
Example 4. Results of Addinq_ Lactobacillus, Whey and
Salmonella
Salmonella senftenberg in feces and caecal contents of
poults treated as in Example 3 is shown in Tables 2 and 3,
respectively. The effect of L. reuteri and whey on the
number of S. senftenberg in feces (droppings) becomes
obvious at 72 h after Salmonella challenge. The data
indicate a synergistic effect when whey and L. reuteri are
added together.
The presence of S. senftenberg in caecal contents is
presented in Table 3. The results show that addition of L.
reuteri and/or whey, but in particular, the combination of
L. reuteri and whey, is effective in reducing the presence
of S. senftenberg in the ceca of these animals. Thus,
whereas 47~ of the control ceca tested positive for S.
senftenberg, none (0%) of the samples tested positive when
fed L. reuteri and whey.
CVO 9?/i?63y PCTII.'S92/0070fd
21~~ r"~~~
_17_
TABLE 2. Logo CFU of S. senftenberg per g feces 72 h pit
challenge.
TREATMENTS
REPLICA CONTROL L.reuteri WHEY WHEY AND
SAMPLES L.reuteri
-_
a . > 10 6 3 4
b. 9 7 8 5
c. > 10 7 6 < 3
d. 10 3 < 3 < 3
TABLE 3. Percent of cecal samples testing positive for S.
senftenberg and L. reuteri 7 days post challenge
TREATMENTS L.reuteri S.sen~,tenberg
CONTROL 0 47
L.reuteri 29 40
WHEY 6 13
WHEY AND 82 0
L.reuteri
Z Positive samples had > 103 CFU/g
Positive samples had < 10 CFU/g
Example 5. Growth of Cold Stressed Poults Fed with L.
reuteri and Whey
Instead of exposing turkey poults to constant
temperature rooms as in Example 1, the temperature in the
pens of cold stressed birds is 90 degrees F for 1 hour,
then 85 degrees G for 2 hours in an on-off cycling far 48
hours after hatch. The temperature is then set back to
normal brooding temperature for the remainder of the
yV'O 92/12639 PCT/US92/00708
_1g_
experiment, normal brooding temperature being 90 degrees F
for the first seven days after hatch, 85 degrees F~from day
7 to day 10, and 75 to 80 degrees after day 10.
Turkey poults are subjected to the following four
treatments , with eight pens of eight birds per pen being in .
each treatment group.
1. Control, no whey, no L. reuteri
2. No whey, L. reuteri
3. Whey, no L. reuteri
4. Whey, L. reuteri
Example 6. Results of Growth of Cold Stressed Poults
Relative wights of poults treated as in Example 5, at
0, 5, 10, 15, and 20 days of age are shown in Table 4. The
beneficial effect of whey becomes evident at day 5, while
the effect of L. reuteri becomes obvious at days 15 and 20.
TABLE 4. Effect of L. reuteri and whey o.n body weight of
turkey poults.
TREATMENTS RELATIVE WEIGHT (PERCENT) AT
Day 0 Day 5 Day 10 Day 15 Day 20
No whey, no 100 200 100 100 100
L.reuteri
No whey, plus 99 98 102 104 105
L. reuteri
Plus whey, no 99 103 101 101 103
L. reuteri
Plus whey, plus 99 103 103 100 105
' L. reuteri
FQr percentage conversion, control weights (no whey, no _L.
reuteri) at each weight day were made equal to 100.
Example 7. Use of Lactobacillus salivarius and
Lactobacillus animalis
L. salivarius subsp. salivarius ATCC type strain No.
11741 and L. animalis ATCC type strain No. 35046 are grown
w0 92/1?639 PCT/US92/00~08
~ :~.~~'~~~
-19-
as in Example 2. Each strain is added individually to feed
as in Example 3. The feed is augmented with whey according
to Example 3. The feed is used to feed chickens and
turkeys to decrease undesirable microbial organisms and
improve poultry weight gain.
Example 8. Use of Multiple Lactobacillus Strains
Strains of the three Lactobacillus species discussed
in Examples 3 and 8 are each added individually, or as a
mixed inoculum to the whey-augmented feed according to
ZO Example 3. The feed containing the three strains is used
to feed turkeys and chickens.
Exam 1e 9. Formation of Pellets
Powdered whey is exposed to compaction at a pressure
of 10-15 lb/in2 to form pellets. The pellets are milled
and sieved to a size which is edible by the birds, for
example, -8, +20 mesh for little pellets and -1/4"; +8 mesh
for larger pellets. Lactobacillus reuteri strain T-1,
11284 or other strains compatible with the intended host
animal species are lyophilized in a cryoprotectant such as
milk or whey and then is mixed in an oil, such as a
sunflower oil-based. drench at a concentration of about 3 x
10'°/g in the oil. The drench may contain trace amounts of
silicon dioxide.
The strains mentioned above have been deposited at the
American Type Culture Collection in Rockville, Maryland.
The pellets of whey are then coated with the
Lactobacillus-containing oil which may be done simply by
pouring the oil-suspension aver the whey pellets so that
there are about 5 x 10T to about 108 cells/g whey. The
survival of the Lactobacillus on the pellets is shown in
the first column of data in Table 5. The whey particles
are then mixed with feed pellets or particles so that the
whey particles comprise 2 - 5~ of the feed by weight, so
that there are 5 x 105 to 106 CFU/g feed mix.
~~'O 92/12639 PC'T/LS92/i)0708
:, .
-20-
Table 5
Time (days) Oil drench In Product Product feed
0 3 x 10r 6 x 108 7 x 106
1 ND 3 x 108 9 x 106
2 ND 2 x 108 4 x 106
3 4 x 10~ 6 x 108 7 x 106
4 ND ND 4 x 106
5 ND 3 x 108 ND
7 3 x 10~ 3 x 108 ND
10 4 x 10~ ND ND
3 x 10~ ND ND
3 x 10~ ND ND
-Example 10. Survival of Lactobacillus in Pellets
A Lactobacillus-oil suspension is prepared as in
15 Example 9. The suspension is then mixed with whey powder
in a concentration of 107 per g whey. The mixture is then
compacted, milled and sieved as in Example 9. Typical
results of survival of the Lactobacillus reuteri in such
pellets is shown in the central data column of Table 5.
20 The survival when such pellets are mixed with feed as done
in Example 9 is shown in the final column of Table 5.
Example 11. Turkeys Fed Pellets
Turkey poults are fed feed and pellets having about
10' CFU L. reuteri/g feed prepared according to Example 10
25 for a period of 10 days. The total number of lactobacilli
found in the bird's cecum is determined for each treatment
as colony-forming units per excised and homogenized cecum.
Solid Lactobacillus selection medium (1.5% agar) as
described in references 2, 5, and 7 is used. The percent .
30 of the colonies which were L. reuteri is determined as
described in international patent application
PCT/US88/01~23 but using L. plantarum as the indicator '
organism. In this test, colonies of lactobaeilli on the
LBS agar medium are overlaid with 10 ml of 1% liquefied
w0 92/12639 PCT/(.~592/00708
agar containing 0.5 M glycerol and a L. plantarum inoculum.
After anaerobic ( GAS-Pack System ) incubation at 37°C for 24
hours, cones of growth inhibition are seen around colonies
that produce reuterin from glycerol. These colonies are
thus identified and enumerated as L. reuteri.
As seen in Table 6, colonization,of the ceca by L.
reuteri is enhanced by the feed treatment as compared to
the control. Only 1/5 of the control birds in the results
shown are positive for L. reuteri, while 4/5 of the treated
birds retain significant numbers of L. reuteri in the
cecum.
. Table 6
CFU per g Ceca ~ Of Birds
Positive For
Total L. reuteri L. reuteri
Lactobacilli
Control birds 9.0 x 108 to 1.5 x 105 to 20~s
1.5 x 10~~ 1.2 x 108
I5 Treated birds 5.0 x lOT to 4.0 x 10' to 80~
3 . 7 x 109 1 , 1 x 109
While the invention has been described with reference
to specific embodiments thereof, it will be appreciated
that numerous variations, modifications, and embodiments
are possible, and accordingly all such variations,
modifications, and embodiments are to be regarded as being
within the spirit and scope of the invention.
BEST MODE FOR CARRYING OUT ~ INVENTION
A formulated product that may be used as an animal ~ '
feed additive includes isolated and identified pure
cultures) of naturally occurring gastrointestinal
microorganisms. Preferably viable cells of Lactobacillus
reuter.i, an oil and whey powder are used. The
Lactobacillus cells may be coated on the surface of whey
»'O 92/12639 PCT/l.'S92/00708
pellets or be contained in the pellets. Preferably
powdered whey is exposed to compaction to form pellets.
The pellets are milled and sieved to a size which is edible
by the birds.
INDUSTRIAL APPLICABILITY
The invention includes a formulated product that may
be used as an animal feed additive. The feed additive
provides Lactobacillus cells to the animal, resulting in
decreased survival of gastrointestinal pathogens and
increased animal weight gain.
~1'O 9?/12639 PCT/L'S92/0070~
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