Studies On Screening, Mechanism Of Action Of Lactic Acid Bacteria For Forage Ensiling

The LAB inoculants could increase LAB count in silage material, so that the fermentation would be dominated by LAB as soon as possible. However, few LAB inoculants are effective for forages with low WSC content. Exploration of mechanism of LAB inoculants application in silage is not deep enough. And suitable conditions are needed for LAB inoculants. In this study, LAB strains were isolated from silage, their effects on fermentation quality, the number of microorganisms, aerobic stability, in vitro gas production kinetics of silage were investigated. To study the effects of growth stage, moisture content, WSC content, ensiling density, storage temperature on silage quality and effectiveness of LAB inoculants, the further screened strains were applied under different conditions. The main results are as follows:(1)15 strains with fast growth rate and high acid productivity were selected from silage:LB, LPL1, LPL2, LPL3, LCL, WH, LP1, LP2, LP3, LCI, LC2, PP1, PP2, EF1, EF2. By physiological, biochemical tests and 16S rDNA sequencing, LB was identified as Lactobacillus brevis, LPL1, LPL2, LPL3, LP1, LP2, LP3 were identified as L. plantarum, LCL, LC1, LC2 were identified as L. casei, PP1, PP2 were identified as Pediococcus pentosaceus, EF1 was identified as Enterococcus faecalis, EF2 was identified as E.faecium.(2) Results indicated the strains improved fermentation quality of Leymus chinensis, alfalfa (Medicago sativa L.), elephant grass (Pennisetum purpureum Schum.) silage, and failed in improving fermentation quality of native grass silage. LB, LPL1, LPL2, LPL3, LCL, and WH improved aerobic stability of Leymus chinensis silage, LB slowed down the aerobic spoilage rate of Leymus chinensis silage. The combination of LC1 or LP1 with cellulase improved in vitro digestibility of elephant grass and alfalfa silage.(3) Environmental conditions affected silage quality and the richness of bacteriaa. Stage of harvest, moisture content, ensiling density and storage temperature had great influence on silage quality. Leymus chinensis harvested earlier (early heading stage) and ensiling at higher density (600 kg m-3) showed better silage quality, and Leymus chinensis harvested earlier (early heading stage) had higher digestibility; better quality alfalfa silage was obtained by proper wilting before ensiling, Lactobacillus showed highest richness and better quality was observed when alfalfa silage was stored at 20℃,30℃;b. Lactic acid bacteria inoculants showed positive effects on silage quality under different conditions. LC1 and LP1 improved the fermentation quality of unwilted and wilted alfalfa silage. LC1 and LP1 improve the fermentation quality of Leymus chinensis silage harvested at both stages, but showed no effect on digestibility. LB, LPL1, LPL2, LPL3, LCL, WH improved fermentation quality of Leymus chinensis silage at two ensiling density levels. High quality Leymus chinensis silage could be obtained by addition of the LAB strains, even though it was harvested at later stage (late heading stage) or ensiled at lower density silage (500 kg m-3),;c. Compared with 20℃,30℃, the richness of Garciella and butyric acid content in alfalfa silage stored at 40℃ were higher; when stored at 20℃,30℃, the richness of Lactobacillus increased by addition of LP1 and LC1.(4) We tried to transmit xylanase gene xynA to LP1 by constructing engineered bacteria. The recombinant plasmid pMG36e-xynA was constructed successfully by inserting xylanase gene xynA in a shutter vector pMG36e.