Isolation Of Lactic Acid Bacteria From Papyriformis And Its Effect On Silage Quality

Broussonetia papyrifera is a kind of plant feed with rich nutrients.Its crude protein,ether extract,trace elements and calcium content are high,which has high feeding value.But the quality of Broussonetia papyrifera silage is not ideal.In this study,the natural fermented Broussonetia papyrifera silage was used as the source of microorganisms to isolate high-quality primary lactic acid bacteria,optimize its culture conditions,and add them as lactic acid bacteria additives into Broussonetia papyrifera silage to study the effects of the primary lactic acid bacteria inoculum on the nutritional components,fermentation quality and microbial diversity of Broussonetia papyrifera silage.The experiment is divided into four parts:1.Isolation and identification of lactic acid bacteriaThe purpose of this experiment was to isolate lactic acid bacteria from natural fermented Broussonetia papyrifera silage,and identify the selected strains by physiological and biochemical identification and molecular biology.Methods:Two strains of lactic acid bacteria（FC1 and LW2）were isolated from Broussonetia papyrifera silage by MRS culture,and the obtained strains were subjected to physiological and biochemical identification,16S rRNA gene sequence identification and homology analysis.Results:The two target strains（FC1 and LW2）were identified as Enterococcus faecalis by physiological and biochemical analysis,16rRNA gene sequence identification and homology analysis,and LW2 was identified as Weisseria intestinalis.2.Optimization of fermentation conditions of lactic acid bacteriaThe aim of this study was to determine the optimal culture conditions of FC1 and LW2 strains.Methods:The effects of liquid volume,seed age,inoculation amount,shaking speed,initial pH and culture temperature on the optical density of FC1 and LW2 were studied by single factor experiment.Then three significant factors were screened from single factor by PB test.Finally,the culture conditions were optimized by response surface methodology.The results were as follows:（1）The optimal culture conditions of FC1 strain were as follows:liquid volume30mL/250mL,seed age 6h,inoculum 2%,shaking speed 180r/min,initial pH 7,culture temperature 37.5℃.Under these conditions,the OD₆₀₀value of FC1 strain was 1.907.（2）The optimal culture conditions of LW2 strain were as follows:liquid volume 50 mL/250 mL,seed age 12 h,inoculum 1.5%,initial pH 6.5,culture temperature 37℃.Under these conditions,the OD₆₀₀value of LW2 strain was 1.954.3.Effect of inoculation of native lactic acid bacteria on fermentation quality of Broussonetia papyrifera silageThis study aimed to study the effect of FC1 and LW2 strains on the fermentation quality of Broussonetia papyrifera silage.Methods:250 mL fermentation tank was used in the experiment.The control group PM0 and lactic acid bacteria inoculation treatment group（FC1 strain treatment group PMF,LW2strain treatment group LW2,commercial Lactobacillus plantarum treatment group PMZ）were set up.The leaves of Broussonetia papyrifera were mixed with FC1,LW2 and commercial Lactobacillus plantarum.Each group was set up three parallel samples to study the effect of inoculation of native lactic acid bacteria on the fermentation quality and nutritional quality of Broussonetia papyrifera silage.The results were as follows:（1）The pH value of mixed silage decreased rapidly 6days before silage,and gradually stabilized.The pH value of PM0 control group was significantly higher than other treatment groups（P<0.05）.During 60 days of Broussonetia papyrifera silage,the LA content in the group with endogenous lactic acid bacteria was higher than that in the PM0 control group and PMZ treatment group.The content of ethyl AA in each lactic acid bacteria additive group increased compared with PM0 group.AA content in PML group was higher than that in other groups at the same time point.In this experiment,only a small amount of BA was detected in the PM0 control group.Only a small amount of PA was formed in each experimental group.Compared with the PM0 control group,the PA values of the other three groups were lower.（2）After 60 days of silage,the DM content of Broussonetia papyrifera silage in each treatment group was slightly lower than that before silage,but the DM content of additive treatment group was slightly higher than that of PM0 group.The CP content in PM0 group was slightly lower than that before silage,but the CP content in each lactic acid bacteria treatment group was slightly higher than that before silage.There was no significant difference in EE content in each treatment group,but it was slightly higher than that before silage.WSC in each treatment group was significantly reduced compared with that before silage,but the WSC content in the treatment group with lactic acid bacteria was lower than that in the PM0 group.NDF content in PML group was significantly lower than that in PM0 group.ADF content in PMZ group was significantly higher than that in PM0 group.4.Effects of inoculation of native lactic acid bacteria on microorganism during dynamic fermentation of Broussonetia papyrifera silageThe purpose of this study was to study the effects of FC1 and LW2 strains on microorganisms in the dynamic fermentation process of Broussonetia papyrifera silage Methods:High-throughput sequencing was used to detect the changes of microorganisms in different treatment groups and different time points during silage.The results were as follows:at the gate level,the dominant bacteria in each group were Thick-walled bacteria,followed by Proteobacteria.At the genus level,the dominant bacteria of PM0 group and PMF were Pediococcus,and decreased with the increase of silage time.The dominant bacteria of PML group and PMZ group were Lactobacillus,and the relative abundance increased with time.In conclusion,adding lactic acid bacteria can improve the fermentation quality of Broussonetia papyrifera silage,change the microbial community structure in the silage process,make lactic acid bacteria quickly become the dominant flora,reduce the number of harmful microorganisms,and improve the fermentation quality of Broussonetia papyrifera silage.