Lactic Acid Bacteria Silage Agents

Silage is a traditional method to keep the quality of feed by anaerobic fermentation. To the corn straw which is consisted mainly of cellulose and hemicellulose, inoculants with lactic acid bacteria and cellulase could increase its nutritional value. Cloning the cellulase gene to lactic acid bacteria and use the recombinant bacteria with cellulolytic activity as the inoculants would have a potential role in ensilage.To express the cellulase gene of Bacillu subtilis Y-106 strain to Lactococcus lactis MG1614. The plasmid pUC18-CMC was digested with PstI/BamHI and the fragment obtained was ligated with the E.coli and L.lactis shuttle plasmid. The ligation was transformed to E.coli and the positive clones were screened on the LB agar plates supplemented with 150ug/mL erythromycin by congo red staining method with 0.2%CMC-Na. The recombinant plasmid from one positive clone was transformed to L.lactis MG1614 by electroporation. The recombinant plasmid was extracted and analyzed by restriction enzymes. Expression of cellulase of L.lactis were performed by the cellulolytic activity determine. The results indicated that pTRKH2, pTRKL2 can not express the activity but pMG36e can, and the cellulolytic activity in extracellular fractions was 0.133 IU/mL.Electrotransformation condition factors were performed to transfer plasmid pTRKH2 into L. lactis MG1614. Cell growth phase, voltage, resistance and incubation time all affected transformation efficiency. The optimum factors are the cell harvested at the middle exponential growth phase, electroporation at 10.0 kV/cm and 400 Ω , 2h cell incubation time. The transformation efficiency goes up to 1.2 × 103 transformants per μ g DNA with those optimum factors, which is higher than optimiz those reported before.Effects of Lactobacillus and cellulase on quality of corn straw were studied onthe experimental lab conditions, the experiment was carried out inoculated lactic acid bacteria, cellulase, lactic acid bacteria and cellulase into wilted corn straw respectively. No inoculants were used control. Applied for ensilage in the laboratory conditions for 35 days. Results showed that the treatment of corn straw with Lactobacillus and cellulase the pH decreased rapidly to 3.8 in the primarily phase and kept stability until the end of the silage process; water-soluble carbohydrate content increased to 74.53mg/g DM, increased by 1.01 times; cellulolytic activity increased to 3.67IU/g DM, increased by 4.46 times; lactic acid content increased to 57.35mg/g DM, increased by 1.27 times; lactic acid bacteria content attained to 9.3 × 109cfu/g DM, increased by 4.47 times. All these indicated that the lactic acid bacteria and cellulase inoculants can improve the quality of corn straw obviously in ensilage.