Effect Of Epiphytic Lactic Acid Bacteria Of Morus Alba Leaves On Its Silage Fermentation

Morus alba L.is a perennial deep rooted deciduous woody plant of the genus Morus inRosales,the whole plant of which is edible and highly valuable for feeding,and can be developed as a novel unconventional feed resource,which will be beneficial to solve the dilemma shortage of high-quality feed in China.In this study,lactic acid bacteria（LAB）were isolated from the leaves of broussonetia papyrifera and morus alba L.,respectively,and identified the screening strains by physiological and biochemical tests and molecular biology,compared the tolerance of mulberry tannins,measure the routine tolerance performance;the above lactic acid bacteria isolate were added as silage additives to mulberry leaves,respectively,to explore the effect of exogenous and epiphytic lactic acid bacteria on the silage quality and microbial diversity of mulberry leaves,with the aim of explore the effect of epiphytic lactic acid bacteria on the silage of feeds.The test is divided into four parts:1、Isolation and identification of LABIn this experiment,LAB were isolated from broussonetia papyrifera and morus alba L,and the strains were identified by physiological,biochemical and molecular biological characteristics,the tolerance of mulberry tannin and temperature of the screened strains was compared.The results showed that:two strains of lactic acid bacteria（GPP and SPP）were isolated from different leaves,GPP was isolated from broussonetia papyrifera,SPP was isolated from morus alba L;Among of them,the tolerance of SPP to mulberry tannin was stronger than GPP,and there was no significant difference in temperature.2、Optimization of culture conditionsThe purpose of this experiment was to compare the optimal culture conditions of GPP and SPP strains by single factor test and response surface optimization.Firstly,the optimal conditions of different influencing factors were determined by single factor test,then,the inoculation time,medium p H and shaker speed were determined as the key influencing factors by PB test design,finally,the optimal culture conditions were determined by BBD experiment design.The results showed that the optimal culture conditions were as follows:the strain GPP was culture time 8.34 h,culture medium p H 6.6 and shaker speed 142.01r/min;The strain SPP was culture time 8.5 h,culture medium p H 6.5 and shaker speed 144.78r/min,indicating that there was no obvious difference in the optimal culture conditions of Pediococcus pentosaceae from different sources.3、Effect of different sources of Pediococcus pentosaceae on the chemical composition and fermentation quality of mulberry leavesThe control group CK（without LAB）,the treatment group T1（with GPP）and the treatment group T2（with SPP）were set up,with 3 replicates in each group.According to the above group design,mulberry leaves were silage respectively,and samples were taken at different times to detect the effects of different sources of LAB on mulberry leaves silage fermentation.The test results show that:（1）、Nutrient Composition Changes:After 5 days of silage,the DM and CP content of the treatment group were significantly higher than the CK group（P<0.05）,but the ADF was significantly lower than CK group（P<0.05）;the DM in the T2 group were significantly higher than T1group after 30-60 days of silage（P<0.05）;the CP content in T2 group was significantly higher than T1 group from 3-60 days（P<0.05）.the DNF in T1 and T2 groups were significantly lower than CK group starting from the 7 and 3 day of silage,respectively（P<0.05）;the NDF of T2 group was significantly lower than T1 group at 3-60 days（P<0.05）.the WSC in the treatment group were significantly lower than CK group in the whole silage period（P<0.05）,with the WSC content in the T2 group significantly lower than T1 group during the 5-60 days of silage（P<0.05）.With the silage progressed,the content of DM,CP,NDF,ADF,and WSC decreased in all groups.（2）、Changes in fermentation quality:At each period of silage,the p H and NH₃-N content of the treatment group were significantly lower than CK group（P<0.05）,but the LA and AA were significantly higher than CK group（P<0.05）.In the 1-7 days of silage,the p H of the T2 group was significantly lower than T1group（P<0.05）,while the LA content was significantly higher than T1 group（P<0.05）.The AA content of the T2 group was significantly lower than T1 group from the 3 days（P<0.05）,and the NH₃-N content of the T2 group was significantly lower than T1 group from the 3-60days（P<0.05）.With the silage progresses,the p H of all groups tend to decrease to stabilize,while the LA,AA,and NH₃-N content of each group gradually increase.4、Influence of different sources of Pediococcus pentosaceae on the community structure of silage microorganisms of mulberry leavesThe aim of this experiment was to explore the effects of Pediococcus pentosaceae from different sources on the microbial community structure during the dynamic fermentation of mulberry leaf silage.The results showed that:The Shannon index of T1 and T2 groups was significantly lower than CK group in the whole silage period（P<0.05）,and T2 group was significantly lower than T1 group（P<0.05）at 3-7 days.With the progress of silage,the Shannon index of each group showed a decreasing trend.According to the principal component analysis（PCA）,the T2 group can adapt to the mulberry silage environment faster than T1 group and quickly reach the dynamic balance of microbial community structure.At the phylum level,all groups were dominated by Firmicutes and Proteobacteria,and the relative abundance of Firmicutes rapidly increases and reaches a relatively stable state in the early stage of silage;In the CK group,although the relative abundance of Firmicutes has been increasing,Proteobacteria has always been the dominant phylum;In the treatment group,Firmicutes remained the first dominant phylum,while the relative abundance of Firmicutes in the T2 group gradually decreased from significantly higher than T1 group.At the genus level,the relative abundance of Lactobacillus,Pediococcus,and Kosakonia in the treatment group was significantly higher than CK group at different silage stages;The T2 group was higher than T1 group during the1-15 days of silage,while the Kosakonia was higher than T1 group in the whole silage period.The relative abundance of pantoea and Shigella genera in the treatment group was lower than CK group during the same silage period;At the same time,the Shigella genera in the T2 group was lower than T1 group.The results showed that:adding lactic acid bacteria from different sources could improve the fermentation quality of mulberry leaf silage,but adding homologous Pediococcus pentosaceae was more beneficial to the preservation of mulberry leaf nutrients,improved fermentation quality,and more effectively improved the microbial community structure of mulberry leaf silage.