Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1999-07-09
2002-12-03
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S267000, C426S062000
Reexamination Certificate
active
06489158
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the silage process, to microorganisms, and use of the same in treating animal feed and silage to enhance aerobic stability, thus preventing spoilage.
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 develop 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 heat 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.
Management (compaction, unloading rates) largely affects 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. Silage inoculants containing principally homofermentative lactic acid bacteria have become the dominant additives in many parts of the world. Their function is to promote rapid and efficient utilization of a crop's water soluble carbohydrates resulting in intensive production of lactic acid and a rapid decrease in pH. Inoculants also reduce aerobic spoilage and improve animal performance.
Several problems, however, with lactic acid bacteria inoculants have been encountered. These problems primarily include failure to dominate fermentation and failure to inhibit adverse microbial activity. Other problems associated with lactic acid bacteria inoculants include infection by phage, failure to grow well on certain crops, bacteria not being viable at the time of application, and the epiphytic nature of the lactic acid bacteria population. Because these types of homofermentative lactic acid bacteria inoculants do not always prevent or reduce undesirable microbial activity, several new approaches have been tried.
A review of the silage process and the use of inoculants can be found in FMS Microbiology Rev. 19 (1996) 53-68, Weinberg, ZNG., and Muck, R E, “New trends and opportunities in the development and use of inoculants for silage”, the disclosure of which is incorporated herein by reference.
The concept of heterofermentative lactic acid bacteria in an inoculant has gained recent favor. The idea is that increased levels of undissociated volatile fatty acids, such as acetate, may inhibit other microbes that initiate aerobic deterioration. Heterofermenters have the ability to convert lactic acid to acetic acid in the presence of oxygen, and the acetate produced may inhibit other deleterious organisms. With such a mechanism, one-third of the lactic acid dry matter consumed will be lost as carbon dioxide. However a small loss of 1% or perhaps up to 2% dry matter may easily offset much larger losses by aerobic microorganisms. Concerns with heterofermentative lactic acid bacteria include effects on animal performance as well as the identification of appropriate strains useful for the procedure. Different strains of even the same species do not have identical properties and vary in their fermentation characteristics.
PCT publication WO 97/29644 discloses a single strain of
Lactobacillus buchneri
(NCIMB 40788) which was found to inhibit the growth of spoilage organisms in the storage of silage. Other attempts to identify heterofermentative organisms for silage inoculants have included (Wyss et al., 1991, “Einfluss von Luftstress und die Wirkung von spezifishen Zusatzen anf die arobe Stabilitat von Grasswelksilagen”, Wirschaftseigene Futter, 37: 129-141), which used an inoculant comprising lactate and propionate producing organisms in wilted grass silage. Weinberg et al. (1995), “The effect of a propionic acid bacterial inoculant applied at ensiling, with or without lactic acid bacteria on the aerobic stability of Pearl-Millet and maize silages”,
J. Appl. Bacteriol
., 78:430-436 disclosed the use of
Propionibacterium shermanii
in millet, corn, sorghum, and wheat silages. Propionic acid was produced only in a wheat silage in which the pH decline was delayed and thus aerobic stability was improved. In all other silages the pH decline was rapid and the propionic acid bacteria could not proliferate.
Another attempt included select strains of
Serratia rubidaea
and
Bacillus subtilis
along with
L. plantarum
. When used in bale grass silages the number of molds decreased significantly. Some improvement was also observed in high moisture ear corn. (Moran et al., (1993), “The development of a novel bacterial inoculant to reduce mold spoilage and improve the silage fermentation in big bale silage. In:
Silage Research
1993
, Proceedings of the Tenth International Conference on Silage Research
(O'Kiely, P., O'Connell, M. and Murphy, J., Eds.) pp. 85-86, Dublin City University, Ireland). A similar composition to that for bale grass silage was developed for wheat silage which added Pediococcus strains to the composition. Pediococcus is capable of fermenting pentose sugars which result from hemicellulose hydrolysis in wheat silages. In a single trial with wheat silage, no improvement in the aerobic stability was observed.
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 imp
Hendrick Carol A.
Hoganson Dean A.
Platt Nancy J.
Ruser Barbara G.
Patterson Jr. Charles L.
Pioneer H-Bred International, Inc.
Pioneer Hi-Bred International , Inc.
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