Reinforcement of Silage-Making Lactobacilli and Their Utilization

青贮料是一种利用乳酸发酵的可储存饲料。开发一种适应日本西南部气候条件的青贮料接种剂，如九州和冲绳。我们在高温条件下分离到了多株产乳酸能力较高的同种发酵乳酸菌。(1)通过常规方法和部分16S rRNA基因、磷酸果糖激酶基因和磷酸果糖激酶基因的测序，将分离到的菌株分为植物乳杆菌、戊糖乳杆菌、干酪乳杆菌和鼠李糖乳杆菌。噬菌体方法也是有效的，因为它们容易和快速地分类。(2)77个青贮料样品中有25%含有噬菌体，其中大多数是感染植物乳杆菌的，其次是干酪乳杆菌。青贮饲料中的噬菌体污染导致了青贮料品质的恶化。(3)所分离的噬菌体抗性质粒pLKS(2.1kb)为Rheta复制型。PLKS的抗噬菌体活性可能是由于其复制因子与噬菌体的相似性所致。(4)将pLKS与pUC119连接，构建了乳杆菌-大肠杆菌穿梭载体pUCLSEm。(5)从堆肥中分离到一株地衣芽孢杆菌161号菌株，该菌株具有较高的硝酸还原酶活性。本试验用来消除青贮料中的NO^-__。此外，还克隆了与E.ColiNarh有显著同源性的硝酸还原酶基因Narh。(6)利用具有抗噬菌体活性的载体pUCLSEm构建了含有α-淀粉酶基因、pFK和NarH基因的植物乳杆菌和干酪乳杆菌的重组菌株。

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.