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Application of molecular biology in the evaluation of Lactobacillus plantarum strains as silage inoculants
Duffner, Fiona
Methods to distinguish between strains of Lactobacillus plantarum were investigated with the aim of analysing strain heterogeneity in grass silage. Chromosomal restriction endonuclease patterns, rDNA fingerprints and plasmid profiling were applied to isolates from five different well preserved grass silages and a number of dominant strains were identified. Ribotyping proved to be a most useful technique for strain differentiation using restriction enzymes EcoRl and BarriHl to achieve optimum results. Plasmid profiling complemented the information obtained from ribotyping while chromosomal restriction endonuclease analysis proved to be too cumbersome for routine use. Having demonstrated that L. plantarum effected improved forage preservation when applied at an inoculation rate of lxlO6 cfu/g grass, a number of L. plantarum strains were investigated for their suitability as grass silage inoculants. A novel assay was designed to assess the competitiveness of each L. plantarum strain when co-inoculated individually with the internal standard strain, L. plantarum DCU101. The plasmid based strain specific DNA probe, pGBlOO, was used to enumerate the proportion of L. plantarum DCU101 in each treatment over the fourteen day ensilage period. The results demonstrated that competitiveness and dominance do occur within the silo and the most competitive strain, L. plantarum B2, was used in further silo trials to establish its usefulness as a bacterial silage inoculant. The PCR amplification of the VI and V6 variable regions of 16S rRNA genes from L. plantarum was investigated for its usefulness in obtaining species and genus specific probes. Direct sequencing from PCR was investigated but failed due to the small size of the PCR fragment (about lOObp) which resulted in its rapid renaturation. The cloned PCR amplified VI and V6 regions from three strains of L. plantarum were sequenced and analysed using hybridisation experiments and computer assisted alignments with sequences extracted from the GenBank and EMBL databases. On alignment of the VI and V6 regions of thirty species of Lactobacillus and a variety of Gram-negative and Gram-positive genera, three L. plantarum specific oligonucleotide sequences were identified. The specificity of these selected oligonucleotides was confirmed using the BLAST alignment programme.
Keyword(s): Biotechnology; Grass research; Molecular biology; Silage Fermentation
Publication Date:
Type: Other
Peer-Reviewed: Unknown
Language(s): English
Contributor(s): O'Connell, Michael
Institution: Dublin City University
Citation(s): Duffner, Fiona (1993) Application of molecular biology in the evaluation of Lactobacillus plantarum strains as silage inoculants. PhD thesis, Dublin City University.
Publisher(s): Dublin City University. School of Biotechnology
File Format(s): application/pdf
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First Indexed: 2013-07-25 05:14:27 Last Updated: 2019-02-09 06:34:09