Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
2000-11-03
2002-06-11
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S093480, C426S052000, C426S053000, C426S056000, C426S332000, C426S335000, C426S532000, C435S042000, C435S139000, C435S252900, C435S253600, C435S857000
Reexamination Certificate
active
06403084
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to the silage process and to microorganisms and use of the same in treating animal feed and silage to enhance aerobic stability of the same.
BACKGROUND OF THE INVENTION
The ensiling process is a method of moist forage preservation and is used all over the world. Silage accounts for more than 200 million tons of dry matter stored annually in Western Europe and the United States alone. The concept involves natural fermentation, where lactic acid bacteria ferment water soluble carbohydrates to form organic acids under anaerobic conditions. This causes a decrease in pH, which then inhibits detrimental microbes so that the moist forage is preserved. The process can be characterized by four different phases.
Upon sealing in the storage unit, the first phase is aerobic, when oxygen is still present between plant particles and the pH is 6.0 to 6.5. These conditions allow for continued plant respiration, protease activity and activity of aerobic and facultative aerobic microorganisms.
The second phase is fermentation, which lasts several days to several weeks after the silage becomes anaerobic. Lactic acid bacteria grow and become the primary microbial population thereby producing lactic and other organic acids, decreasing the pH to 3.8 to 5.0.
The third phase is stable with few changes occurring in the characteristics of the forage so long as air is prevented from entering the storage unit.
The final phase is feedout when the silage is ultimately unloaded and exposed to air. This results in reactivation of aerobic microorganisms, primarily yeast, molds, bacilli and acetic acid bacteria which can cause spoilage.
Aerobic instability is the primary problem in silage production. Even before storage units are open for feedout, silage can be exposed to oxygen because of management problems (i.e., poor packing or sealing). Under these types of aerobic conditions, rapid growth of yeast and mold cause silage to and spoil, decreasing its nutritional value.
Aerobic instability can be a problem even in inoculated silage that has undergone what would traditionally be considered a “good” fermentation phase, namely a rapid pH drop, and a low terminal pH. The yeast which contribute to instability in these conditions may be those which are tolerant of acid conditions and can metabolize the lactic acid produced by lactic acid bacteria during fermentation.
Management techniques that can be used to help prevent this condition involve using care to pack the silage well during the ensiling process and, also, using care in removing silage for feeding to minimize the aeration of the remaining silage.
The susceptibility of silage to aerobic deterioration is determined by physical, chemical, and microbiological factors. Management (compaction, unloading rates) largely effects the movement of oxygen into silage. During feedout, air can penetrate 1 to 2 m behind the silage face so that exposure to oxygen is prolonged. Fermentation acids and pH inhibit the rate of microbial growth but spoilage rates are affected also by microbial numbers and the rate of aerobic microbial growth on available substrates.
It is possible to use both chemical and biological additives in making silage to promote adequate fermentation patterns especially under sub-optimal conditions. Biological additives comprise bacterial inoculants and enzymes. Bacterial inoculants have advantages over chemical additives because they are safe, easy to use, non-corrosive to farm machinery, they do not pollute the environment and are regarded as natural products.
Lactic acid bacteria (LAB) are present as part of the normal microflora on growing plants. LAB can be classified as one of two types depending upon their primary metabolic end products; homofermentative which produce only lactic acid from the metabolism of glucose and hetrofermentative which produce lactic acid, ethanol, acetate and CO
2
. The occurrences of these types are quite variable in both type and number, crop to crop and location to location. There appears to be some dependence upon the environmental conditions but in general it appears that the ensiling process is dominated by homfermentative LAB.
Nilson (
Arch Microbiol
. (1956) 24: 396-411) found that the predominant LAB in silage are Streprococci and Lactobacilli with
L. plantarum
being the most frequent species. Gibson et al,
J. Gen. Micro
. (1958) 19: 112-129) reported that
L. plantarum
and
L. acidophilis
were the dominant component of the homofermentative flora. Beck (
Landwirtschaftliche Forschung
. (1972) 27: 55-63) showed that even in grass silage where the epiphyte population was dominated by heterofermentative LAB, by day four of the ensiling process 85% of the organisms were homofermentative. Langston et al. (USDA
Technical Bullitin
No. 1187 (1958)) has shown that the 69% of the isolates in mature silage were homofermentative. A shift is sometimes noted toward homofermentative LAB in mature silage owing to their own tolerance to low pH and high acetate concentrations. Szigeti (
Acta Almentaria
. (1979) 8:25-40) found that the LAB flora at extremely low pH consisted mainly of
L. plantarum
and
L. brevis.
Grazia and Suzzi (
J. Appl. Bacteriol
. (1984) 56: 373-379) have shown that a strong sensitivity to pH 3.6 was observed among the herofermentative LAB. The lack of pH tolerance coupled with the predominance of homofermentative LAB early in ensiling would suggest against inoculation of silage with a combination of homofermentative and homofermentative LAB.
The ensiling process is a complex one and involves interactions of numerous different chemical and microbiological processes. Further, different silages and different methods of ensiling present a variety of different needs. As can be seen a need exists in the art for further improvement in compositions and methods to improve the aerobic stability of silage. The present invention provides novel strains of
L. buchneri
and superior combinations of homofermenters and heterofermenters for use as silage inoculants.
SUMMARY OF THE INVENTION
The present invention provides surprisingly effective, isolated and purified combinations of the homofermentive lactic acid bacteria
L. plantarnu
with the heterofermentive lactic acid bacteria
L. buchneri
or
L. brevis
for use as silage inoculants. The silage inoculants provided herein provide sufficiently low pH to assure adequate preservation of ensiled forage while retaining the aerobic stability enhancement imparted by the heterofermentive bacteria. The silage inoculant has a ratio of viable homofermentive bacteria to heterofermentive bacteria of about 1:5 to about 1:15. In optional embodiments the preferred ratio is 1:10. In additional embodiments
L. buchneri
, such as strains LN1391, LN4637, or LN4750, is provided. In some embodiments the silage inoculant will comprise a viable culture of
Enterococcus faecium.
The present invention also provides methods of treating animal feed or silage with the silage inoculant of the present invention, as well as the treated animal feed or silage itself. Often, the animal feed or silage will be whole plant corn silage (WPCS) or high moisture corn (HWC). The present invention also provides a method of improving animal performance by feeding the inoculated animal feed or silage. Containers comprising the silage inoculant of the present invention and a carrier are also provided.
Definitions
Units, prefixes, and symbols may be denoted in their SI accepted form. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range.
As used herein, “functional mutant” means a bacterial strain directly or indirectly obtained by genetic modification of, or using, the referenced strain(s) and retaning at least 50% of the activity of a control silage using the referenced strain. The genetic modification can be achieved through any means, such as but not limited to, chemical mutagens, ionizing radiation, transposon based mutagenesis, or via conjugation, transduction,
Chan Russell Kuo-fu
Dennis Scott
Harman Elizabeth K.
Rutherford William
Smiley Brenda
Lankford , Jr. Leon B.
Pioneer Hi-Bred International , Inc.
Quine Intellectual Property Law Group P.C.
Ran, Esq. David B.
Srivastava Kailash C.
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