Screening Of Lactic Acid Bacteria In Broussonetia Papyrifera Leaves Silage And Effect On The Composition Of The Microflora And Fermentation Quality

Silage is a method of using anaerobes to preserve fresh fodder crops.Silage can effectively preserve high nutritional value.Broussonetia papyrifera has the characteristics of high protein,strong stress resistance and easy to obtain,so it has been developed as a new woody feed resource in recent years.However,the silage of Broussonetia papyrifera could be awful,easy to spoil,and produce peculiar smell,so it is especially necessary to add lactic acid bacteria to regulate the microflora of silage and improve the fermentation quality of silage.Because the ecological adaptability of lactic acid bacteria screened from different silage materials is different,screening lactic acid bacteria from Broussonetia papyrifera silage will be more competitive.At present,there are few reports on the screening and application of lactic acid bacteria in Broussonetia papyrifera leaves,so it is very important to screen the lactic acid bacteria suitable for the environment of Broussonetia papyrifera leaves silage.The specific results are as follows:(1)The main organic acids in silage include lactic acid,acetic acid,propionic acid and butyric acid,and their content determines the quality of silage.In order to develop an effective method for the determination of four organic acids(acetic acid,propionic acid,butyric acid and lactic acid)in Broussonetia papyrifera leaves silage,high performance liquid chromatography-mass spectrometry(HPLC-MS)was used to quantify the peak area of mass spectra derived from organic acids,2-hydrazinoquinoline(HQ)is used as a derivative agent.The results showed that the derivation efficiency is the best when the organic acid sample uses acetonitrile as the sample solvent and the water content is controlled at 10%.The optimized derivation conditions are as follows: 4 m M triphenylphosphine(TPP),3 m M2,2-Dipyridyldisulfide(DPDS),3 m M HQ,50 ℃,20 min,which can realize the accurate quantification of organic acids.The linear ranges of acetic acid,propionic acid and butyric acid(0.0001%～0.005%)and lactic acid(0.01%～0.5%)were determined under the non-interference of four organic acids.The precision(RSD ≤7.45%)and stability of the method are good(RSD ≤ 6.72%),which provides a method basis for subsequent detection of silage samples(2)In order to screen the lactic acid bacteria suitable for Broussonetia papyrifera leaves silage,four batches of Broussonetia papyrifera leaves were silaged in two years,the last isolated strains were inserted into each generation of silage,and the dominant strains were isolated when the silage was terminated.Using plate separation technology and 16 S r DNA sequence analysis,the main microbial strains isolated and identified were Lactobacillus plantarum,Lactobacillus brevis,Enterococcus faecium,Lactobacillus coryniformis,Lactobacillus crustorum.The characteristics of microbial community in each generation of leaves silage system were analyzed by 16 S r DNA high-throughput sequencing technology.It was found that Lactobacillus and Enterococcus were the main dominant genera.(3)Through two years and four batches of silage fermentation experiments,it is found that due to the changes of seasonal temperature,humidity and material nutrition,Lactobacillus inoculant can not always become the dominant bacteria,but as long as it is connected with Lactobacillus inoculant,it can ensure that the proportion of Lactobacillus is more than 43%,and the silage effect is stable,indicating that the Lactobacillus inoculant is effective.(4)16S rDNA high-throughput sequencing technology was used to evaluate the competitive ability of bacterial agents in the bacterial community,Lactobacillus brevis E4,E5 and E6,Lactobacillus plantarum D3 and F3 have good competitive ability and can be used as candidates for bacterial agents.Due to the existing culture methods,some dominant strains can not be isolated and propagated,which affects the competitiveness and adaptability of bacterial agents in the flora,which is a key breakthrough problem.(5)In order to evaluate whether the addition of lactic acid bacteria to Broussonetia papyrifera leaves can improve the fermentation quality of silage,the fermentation quality of the same batch of Broussonetia papyrifera leaves silage was analyzed,yeast and mold were not detected in silage process,It was found that lactic acid bacteria could reduce the p H of silage,increase the ratio of lactic acid to total acid,reduce the number of Enterobacteriaceae and the content of butyric acid,preserve more nutrients in Broussonetia papyrifera leaves silage.In general,the inoculation of lactic acid bacteria in the silage of leaves ensured the high proportion of Lactobacillus,and the existence of pathogenic bacteria was not found,which ensured the basic quality of silage.The results indicated that the silage with lactic acid bacteria was safe to feed in terms of microorganism analysis.However,it is clear that there are still many problems that need to be further studied so that high quality silage bacterials can be produced.