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Eighteen strains of Pediococcus acidilactici were screened in the laboratory for their potential as silage inoculants. The grass silage isolate, P. acidilactici G24, was found to be the most suitable, primarily on the basis of its short lag phase. Inoculation of grass of water soluble carbohydrate content greater than lOOg/kg DM with P. acidilactici G24 at a rate of 106/g forage consistently aided silage preservation, as indicated by a more rapid rate of lactic acid production and pH decrease, and silage with a lower pH, a lower ammonia nitrogen content and a higher level of crude protein than uninoculated controls. Furthermore, when compared to inoculants consisting of Lactobacillus plantarum or mixtures of L. plantarum and P. acidilactici, P. acidilactici G24 initiated a more rapid rate of pH decrease and maintained the same silage pH up to 60 days after ensilage, implying that P. acidilactici G24 is superior to these more traditional inoculants. When tested on grass of water soluble carbohydrate content less than 60g/kg DM, P. acidilactici G24 failed to aid preservation. A second inoculant was therefore developed to cope with low water soluble carbohydrate conditions where starch is available as an alternative source of water soluble carbohydrate. This situation is frequently encountered in the ensilage of alfalfa. While no amylolytic strain of lactic acid bacteria suitable as a silage inoculant could be isolated, the amylase enzyme of Lactobacillus amylovorus was found to be well adapted to typical ensiling conditions. A region of DNA coding for the L. amylovorus a-amylase enzyme was therefore cloned and integrated into the Cbh gene of the competitive inoculant strain, L. plantarum LP80. A Lactobacillus host strain was chosen in order to comply with regulations governing the release of genetically engineered organisms into the environment. The amylolytic derivative, L. plantarum LPGAFA5, secreted 49% of the amylase secreted by L. amylovorus, stably maintained amylase activity for 50 generations under non-selective conditions and displayed no reduction in growth rates on glucose based media compared to the parent strain, L. plantarum LP80. |
