IMMUNOSTIMULATING AND GROWTH-ENHANCING PREPARATIONS

PREPARATIONS IMMUNOSTIMULANTES ET ACCELERATRICES DE CROISSANCE

English Abstract

A process for obtaining a feed additive material in which a gram-positive
bacterium is utilized to effect incorporation and in-vivo modification of high
concentrations of nucleic acid material. The nucleic acid material is
recovered by
either part of the recovered cellular biomass after lysis or as a part of the
cellular
biomass after lysis concomitantly with the concentrated cell-free broth. The
lysed
isolated or unseparated biomass if useful for low level inclusion in animal
feeds.

Claims

4
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of formulating a thermostable, growth-enhancing feed
formulation, comprising:
providing a source of gram-positive bacteria;
providing culture media containing at least one of yeast RNA,
oligonucleotides,
RNA precursors or derivatives of RNA selected from AMP, CMP, GMP, IMP, UMP,
adenine, cytosine, guanine, thymine, uracil, inosine, adenosine, cytidine,
guanosine,
uridine, thymidine, orotic acid and salts thereof;
fermenting said bacteria in said culture media to provide a cell-free broth
fraction and a cellular fraction;
recovering said cellular fraction;
disrupting cells in said cellular fraction; and
combining said cellular fraction with feed material to form a growth-enhancing
feed material.
2. The method as set forth in claim 1, wherein said bacteria are
selected from the group consisting of Lactobacillus casei ATCC 7469,
Micrococcus
luteus ATCC 4696.
3. The method as set forth in claim 1, wherein said RNA fractions
comprise at least one of concentrated yeast fractions, yeast extract material,
RNA
derivatives selected from AMP, GMP, IMP, UMP, adenine, uracil, inosine,
guanine and
orotic acid.
4. The method as set forth in claim 1, wherein said culture media includes
at least two of whey, whey permeate, soy peptone, cottonseed peptone, sodium
acetate, ammonium citrate, magnesium sulfate, manganese sulfate, cobalt
carbonate,
surfactants, cane molasses, beet molasses, potassium phosphate, copper
carbonate
and mixtures thereof.
5. The method as set forth in claim 1, wherein said formulation contains
between 0.02% and 0.2% dry basis by weight bacteria.

5
6. The method as set forth in claim 1, further including the step of
processing said bacterial cellular fraction by treatments selected from the
group
comprising: high pressure cellular disruption, bead milling and lysozyme
treatment.
7. The method as set forth in claim 1, further including the steps of
concentrating said broth fraction by evaporation and subsequently treating
said
bacterial fraction treated by a treatment selected from the group consisting
of high
pressure cellular disruption, bead milling or lysozyme treatment.
8. The method as set forth in claim 1, wherein said culture media has a pH
of between 6.0 and 8Ø
9. A method of formulating a thermostable, growth-enhancing feed
additive, comprising:
providing a source of gram-positive bacteria;
providing culture media containing yeast RNA, oligonucleotides, RNA
precursors or derivatives of RNA selected from AMP, GMP, IMP, UMP, adenine,
uracil,
inosine, uridine, thymine, guanine and orotic acid. The culture media includes
at least
two of whey, whey permeate, soy and or cottonseed peptones, sodium acetate,
ammonium citrate, magnesium sulfate, manganese sulfate, cobalt carbonate, Span
TM
surfactants, Tween R surfactants, cane and/or beet molasses, potassium
phosphate,
copper carbonate and mixtures thereof.
fermenting said bacteria in said culture media to provide a broth fraction and
a
bacterial cellular fraction; and
recovering said bacterial cellular fraction, subjecting to hydrolysis, and
combining resultant thermostable cellular fraction with feed material to form
a growth-
enhancing feed material.
10. The method as set forth in claim 9, wherein said bacteria include
Lactobacillus casei ATCC 7469, Micrococcus luteus ATCC 4696.
11. The method as set forth in claim 9, wherein said RNA compounds
comprise at least one of the following: concentrated yeast fractions, yeast
extract
material, RNA, oligonucleotides, RNA precursors or derivatives selected from
the

6
group comprising of AMP, GMP, IMP, UMP, adenine, uracil, inosine, uridine,
thymine,
guanine and orotic acid.
12. The method as set forth in claim 9, wherein said culture media includes
at least two of whey, whey permeate, soy and/or cottonseed peptones, sodium
acetate, ammonium citrate, magnesium sulfate, manganese sulfate, cobalt
carbonate,
Span TM surfactants, Tween R surfactants, cane and/or beet molasses, potassium
phosphate, copper carbonate and mixtures thereof.
13. The method as set forth in claim 9, wherein said final compounded feed
formulations contain from between 0.02 and 0.2% by weight of the thermostable
dry
bacterial product.
14. The method as set forth in claim 9, further including the step of
processing said bacterial cellular fraction by individual or combined
treatments selected
from the group comprising: high pressure cellular disruption, ultrasonic
disruption, bead
milling, autolysis, or lysozyme treatment.
15. The method as set forth in claim 9, further including the step of
concentrating said culture media by evaporation and subsequently treating said
concentrated media fraction by individual or combined treatments selected from
the
group comprising: high pressure cellular disruption, ultrasonic disruption,
bead milling,
autolysis or lysozyme treatment.
16. The method as set forth in claim 9, wherein said culture media has an
initial pH of between 6.0 and 8.0 after inoculation.
17. The method as set forth in claim 9, wherein said cellular fraction is
added to final animal feed in a dry form.
18. The method as set forth in claim 9, wherein said cellular fraction is
added to final animal feed in a wet form.

7
19. The method as set forth in claim 9, wherein RNA compounds are
present at the onset of cultivation.
20. The method as set forth in claim 9, wherein RNA compounds are added
after onset of cultivation.
21. The method as set forth in claim 9, wherein RNA compounds are added
continuously during cultivation.

Description

CA 02332749 2001-O1-26
IMMUNOSTIMULATING AND GROWTH-ENHANCING PREPARATIONS
The present invention relates to immunostimulating and growth-enhancing
thermostable preparations and more particularly, the present invention relates
to such
preparations for use in compounded feeds.
One aspect of the present invention is to provide a method of formulating a
thermostable, growth-enhancing feed formulation, comprising:
providing a source of gram-positive bacteria;
providing culture media containing at least one of yeast RNA,
oligonucleotides,
RNA precursors or derivatives of RNA selected from AMP, CMP, GMP, IMP, UMP,
adenine, cytosine, guanine, thymine, uracil, inosine, adenosine, cytidine,
guanosine,
uridine, thymidine, orotic acid and salts thereof;
fermenting the bacteria in the culture media to provide a cell-free broth
fraction
and a cellular fraction;
recovering the cellular fraction;
disrupting cells in the cellular fraction; and
combining the cellular fraction with feed material to form a growth-enhancing
feed material.
A further aspect of the present invention is to provide a method of
formulating a
thermostable, growth-enhancing feed additive, comprising:
providing a source of gram-positive bacteria;
providing culture media containing yeast RNA, oligonucleotides, RNA
precursors or derivatives of RNA selected from AMP, GMP, IMP, UMP, adenine,
uracil,
inosine, uridine, thymine, guanine and orotic acid. The culture media includes
at least
two of whey, whey permeate, soy and or cottonseed peptones, sodium acetate,
ammonium citrate, magnesium sulfate, manganese sulfate, cobalt carbonate,
SpanT"~
surfactants, Tween~ surfactants, cane and/or beet molasses, potassium
phosphate,
copper carbonate and mixtures thereof.
fermenting the bacteria in the culture media to provide a broth fraction and a
bacterial cellular fraction; and
recovering the bacterial cellular fraction, subjecting to hydrolysis, and
combining resultant thermostable cellular fraction with feed material to form
a growth
enhancing feed material.

CA 02332749 2001-O1-26
2
Figure 1 is a flowchart describing the process according to one embodiment of
the invention together with the two variant methods of post fermentation
processing.
In order to formulate an effective preparation for addition to feed material,
a
group of selected strains of gram-positive bacteria were grown in carbohydrate
based
culture media to provide inoculum cultures for subsequent production volumes.
In the
example, the bacteria include Lactobacillus casei and Micrococcus luteus. The
culture
media also contained high concentrations of defined RNA compounds of
fermentative
or synthetic origin, typical examples of which include yeast RNA,
oligonucleotides, RNA
precursors or derivatives of RNA selected from AMP, GMP, IMP, UMP, adenine,
uracil,
inosine, guanine and orotic acid. The culture media includes at least two of
whey, whey
permeate, soy peptone, sodium acetate, ammonium citrate, magnesium sulfate,
manganese sulfate, cobalt carbonate, SpanT"' surfactants, Tween~ surfactants,
cane
and/or beet molasses, potassium phosphate, copper carbonate and mixtures
thereof.
Cultivation may be from six to thirty-six hours.
Subsequent to inoculation preparation, the bacterial inoculum is added in
proportions ranging from between 1 and 20% v/v to production media in the form
of
carbohydrates, sources of nitrogen, minerals and defined RNA compounds of
fermentative or synthetic origin supra. Cultivation may be from twelve to
forty-eight
hours.
At this stage, the cells may be harvested by centrifugation or filtration, the
cells
disrupted by lysozyme reaction, mechanical reaction and/or autolytic reaction
standardized with carriers and dried. The so-formed additive may then be added
to
feed mixtures. As an alternate method, the whole culture slurry may be
concentrated
by known means, prior to lysozyme and/or mechanical and/or autolytic
treatment.
Further, high pressure cellular disruption, ultrasonic disruption, and/or bead
milling may
be employed as suitable alternatives or complements to the lysozyme treatment
and to
cell controlled autolysis..
The resulting slurry of any of the two methods is then standardized by
addition
of any or any combination of the following inert carriers: brewers spent
grains, distillers
spent grains, mineral clays, silica compounds, powdered grain fractions,
starches or

CA 02332749 2001-O1-26
3
dextrins. The resulting mixtures are dried by any of the known methods, such
as
freeze-drying, spray-drying, flash drying, drum drying or fluid bed drying.
Alternatively,
the liquid slurry comprising the bacterial product along with the
standardizing carrier
may be used in its liquid form. A small amount of flavoring agent may
optionally be
added.
Figure 1 illustrates the two reaction schemes in process flow diagram form.
The formulation contains between 0.02% and 0.2% by weight (dry basis) of the
thermostable standardized bacterial product.
Although embodiments of the invention have been described above, it is not
limited thereto and it will be apparent to those skilled in the art that
numerous
modifications form part of the present invention insofar as they do not depart
from the
spirit, nature and scope of the claimed and described invention.

Representative Drawing