CA2332749A1 - Immunostimulating and growth-enhancing preparations - Google Patents

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.

Description

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.

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

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.

Claims (21)

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.

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 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.

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.