Reinforcement of Silage-Making Lactobacilli and Their Utilization

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Silage is a storable feed utilizing lactic fermentation. To develop a silage inoculant adaptable to the climatic conditions of south-west Japan such as Kyushu and Okinawa. we isolated many homofermentative lactobacilli strains with high lactic acid productivity under high temperature conditions. We then examined their properties and improved them to their greatest potential for use as silage inoculant.(1) The strains isolated were classified into L.plantaraum, L.pentosus, L.casei and L.rhamnosus by the usual methods and sequencing of partial 16S rRNA genes and phosphofructokinase genes and phosphofrutokinase gene pfk. A phagetying method was also effective for their easy and quick classification. : (2) 25% of 77 silage samples tested contained phages, most of which were infectious to L.plantaraum followed by L.casei. The phage contamination of silage resulted in the deterioration of quality. : (3) A phage resistant plasmid pLKS (2.1 kb) isolated was a rheta-replicating type. The phage resistant activity of pLKS might be due to the similarity between the replication factors of pLKS and phages. : (4) Lactobacillus-E.coli shuttle vector pUCLSEm was constructed by ligating pLKS to pUC119. : (5) A strain No. 161 belonging to Bacillus licheniformis, which had a high activity of nitrate reductase, was isolated from a compost. This train was tried to use for the elimination of NO^-_ in the silage. Furthermore, nitrate reductase gene narH with significant homology to the E.coli narH was cloned. : (6) The recombinant strains of L.plantaraum and L.casei were constructed with alpha-amylase gene, pfk and narH using vector pUCLSEm which had a phage resistant activity.