Study On Isolation And Identification Of Dominant Lactic Acid Bacteria From Broussonetia Papyrifera Silage And Application For Silage Preparation

Obiective:In this experiment,Broussonetia papyrifera and Broussonetia papyrifera silage were taken as the research objects,the epiphytic lactobacillus attached to them were isolated,16S r DNA identification,acid production rate and growth rate analysis,and then selected the excellent lactobacillus as inoculations,inoculate to Broussonetia papyrifera silage,investigated the effect on fermentation process,aerobic stability,microbial diversity and rumen degradation rate of Broussonetia papyrifera silage,which provided a theoretical foundation for developing lactic acid bacteria in practice.Methods:The traditional microbial culture method and 16S r DNA sequence analysis were used to identify the isolated lactobacillus attached to Broussonetia papyrifera silage.According to physiological and biochemical characteristics,acid production rate and growth rate,use the selected lactobacillus G4,G10and Q5 alone or compound inoculate to Broussonetia papyrifera silage.There were 7 treatments as followed,control treatment（CK）,inoculation of Lactobacillus plantarum（LP）,Lactobacillus brevis（LB）,Enterococcus faecium（EF）,L.plantarum+E.faecium（LPEF）,L.plantarum+L.brevis（LPLB）,L.plantarum+E.faecium+L.brevis（LLE）,the inoculation amount of all treatments was 5×10⁶CFU/g FM（the ratio of compound inoculants is 1:1,1:1:1）,and each treatment has 5 replicates,fermentation lasts for60 days.After 3,7,14,30 and 60 of ensilage,access the nutritional quality,fermentation characteristics and microbial quantity of the Broussonetia papyrifera silage.At the same time,the p H,organic acids and microbial quantity of the silage on the 5th day of aerobic exposure,and the rumen degradation rate of the silage nutrients after 60 days of fermentation were measured at 12 h,24 h and 48 h respectively.Results:（1）A total number of 8 lactobacillus were isolated in experiment,of which strains G4,G7 and G9were identified as Lactobacillus plantarum,G10 and G12 were identified as Lactobacillus brevis,Q3 and Q5 were identified as Enterococcus faecium,and Q7 was identified as Pediococcus pentosaceus.The isolated lactobacillus could grow at 4℃,10℃,30℃and 45℃.It also grow well at 3%and 6.5%Na Cl.7strains could grow or faint grow in the range of p H3.5-p H9.In addition,the strains Q3,Q5 and Q7 gave a positive react of p H3.The characteristics of lactobacillus isolated in Broussonetia papyrifera silage included high acid and high salt resistance,and a wide variety of temperature adaptation range.（2）During the fermentation period,the p H value,dry matter,neutral detergent fiber,crude protein content and the number of lactobacillus,mold and aerobic bacteria in each treatment all showed a downward trend,while the content of ammoniacal nitrogen,lactic acid,acetic acid and propionic acid gradually increased with the extension of fermentation time,the content of acid detergent fiber and crude ash had no obvious change.Among them,except ADF,all treatments can significantly affect the nutrient indexes of Broussonetia papyrifera silage（P<0.05）.LP and LLE treatment significantly reduced the p H value and NH₃-N content of silage（P<0.05）,LP,LB and EF treatments significantly reduced the water soluble carbohydrates content of silage（P<0.05）,and L.brevis alone or compound inoculation（LB,LPLB and LLE treatments）significantly increased the AA content（P<0.05）.Except CK and LP treatments,Mold were not detected after silage for 30 days.After 5 days of aerobic exposure,L.brevis alone or compound inoculation（LB,LPLB and LLE treatments）showed strong aerobic stability.（3）Inoculation of L.plantarum and L.brevis alone or in combination（LP,LB and LLE treatments）could increase the abundance of Lactobacillus and decrease the abundance of Aerococcus in Broussonetia papyrifera silage.E.faecium alone or compound inoculation（EF,LPEF and LLE treatment）could increase the abundance of Enterococcus and decrease the abundance of Lactobacillus.The correlation analysis between fermentation quality and microbial diversity showed that p H was negatively correlated with the abundance of Lactobacillus and positively correlated with the Enterobacter,LA content was positively correlated with the abundance of Lactobacillus and Enterococcus at the 3th and 60th days of silage respectively,and NH₃-N content was positively correlated with the abundance of Aerococcus.（4）Dry matter digestibility,neutral detergent fiber digestibility,acid detergent fiber digestibility,organic matter digestibility of each treatment were improved with the extension of rumen residence time.Among them,the DMD and NDFD of LPEF treatment were always the lowest in all treatments,the ADFD of LP treatment at 12 h and 48 h was significantly higher than CK treatment,and the OMD of each treatment had no significant difference at different times.Conclusion:（1）The combined inoculation of Lactobacillus plantarum,Enterococcus faecalis and Lactobacillus brevis could preserve the content of dry matter and crude protein to the greatest extent,reduce the corruption of protein.（2）Co-inoculation of L.plantarum and L.brevis can maintain the number of lactic acid bacteria during fermentation and within 5 days of opening the bag,improve the aerobic stability of silage.（3）In the combined inoculation of L.plantarum and E.faecalis,E.faecalis limited the growth and reproduction of L.plantarum to some extent.（4）Inoculation of L.plantarum and L.brevis alone or in combination can increase the relative abundance of Lactobacillus and decrease the relative abundance of Aerococcus in Broussonetia papyrifera silage.E.faecium alone or combined inoculation could increase the relative abundance of Enterococcus and decrease the relative abundance of Lactobacillus in silage.（5）Inoculation with L.plantarum alone could improve the degradation rate of ADFD at 12 h and 48 h,while other treatments had no significant effect on rumen degradation rate.