| Broussonetia papyrifera belongs to the mulberry family.It is an unconventional forestry protein feed resource with great development value.Broussonetia papyrifera leaves are rich in crude protein,up to 20% ～ 30%,and rich in amino acids,vitamins,carbohydrates and trace elements.The development of Broussonetia papyrifera as a high-quality unconventional feed source will help to alleviate the shortage of high-quality roughage in China.The purpose of this study was to isolate and screen excellent lactic acid bacteria from silage Broussonetia papyrifera feed,identify them by physiological and biochemical tests and molecular biology,and optimize their fermentation conditions and culture medium.The isolated lactic acid bacteria were added into the silage of Broussonetia papyrifera as Silage Additives to study the effects of the lactic acid bacteria from Broussonetia papyrifera on the fermentation quality and microbial diversity of Broussonetia papyrifera silage,in order to provide a theoretical reference for the preparation of high-quality Broussonetia papyrifera silage.The test is divided into four parts:1.Isolation and identification of lactic acid bacteriaIn this experiment,lactic acid bacteria were firstly isolated from natural silage fermented trees,and physiological and biochemical tests and molecular biology identification were performed to identify suitable lactic acid bacteria strains for silage inoculation of trees.Experimental method: Two strains of lactic acid bacteria were isolated from the silage of Gymnema sylvestre using MRS medium,and physiological and biochemical tests and molecular biology identification were performed.The test results showed that two lactic acid bacteria strains(L1 and L2)were isolated,L1 was Lactobacillus sakei and L2 was Pediococcus pentosaceus.2.Optimization of lactic acid bacteria fermentation conditionsThe purpose of this experiment was to determine the optimal culture conditions and fermentation medium formulations for strains L1 and L2 by a combination of single-factor test,response surface optimization and orthogonal test.The four culture conditions(incubation time,inoculum level,initial p H,incubation temperature)were screened by single-factor test,and then a four-factor,three-level response surface optimization test was designed based on the single-factor test to determine the optimal culture conditions for strain L1: incubation time 8.3 h,inoculum level 2.5%,initial p H=6.15,incubation temperature 37.8℃;the optimal culture conditions for strain L2 The optimal culture conditions for strain L2 were: incubation time of 6.7 h,inoculum level of 2%,initial p H=6.45,and incubation temperature of 37℃.After that,the carbon sources of glucose,sucrose,lactose and maltose were screened by single-factor test,and the best carbon source for strains L1 and L2 was sucrose;the nitrogen sources of peptone,beef powder,yeast powder and urea were screened by single-factor test,and the suitable nitrogen sources for strains L1 and L2 were beef powder and yeast powder with the best effect.After that,a four-factor,three-level orthogonal test was designed on the basis of carbon source and nitrogen source to determine the optimal medium formulation for the two strains.The best formulation of medium for strain L1 was obtained as follows: sucrose 2%,beef meal 1.5%,yeast powder 1.5%,dipotassium hydrogen phosphate 0.3%,Tween-80 0.1%,magnesium sulfate 0.058%,sodium acetate 0.5%,manganese sulfate 0.025%;the best formulation of medium for strain L2 was obtained as follows: sucrose 2%,beef meal 1.5%,yeast powder2.0%,dipotassium hydrogen phosphate 0.3%,tween-80 0.1%.Tween-80 0.1%,magnesium sulfate 0.058%,sodium acetate 0.5%,manganese sulfate 0.025%.3.Effect of Lactobacillus constructus source on the nutrient composition and fermentation quality of constructus leaf silageThe aim of this experiment was to investigate the effect of Lactobacillus constructus-derived strains L1 and L2 on the nutrient composition and fermentation quality of leaf silage of L.constructus,with reference to commercial Lactobacillus plantarum L3.A control group and a lactobacillus followed by treatment group were set up to silage the leaves of L.papyrifera,and the nutrient composition and fermentation quality of each group were determined after 60 d of silage,as well as the p H at different time points.the test results showed that: 1.the DM content of the CK group was significantly lower than that of each lactobacillus added test group(P<0.05),but the difference between the test groups was not significant(P>0.05);the crude protein(CP)of each test group content in all experimental groups was significantly higher than that in the CK group(P<0.05),and the CP content in the L1 and L2 groups was significantly higher than that in the L3 group(P<0.05),but the difference between L1 and L2 was not significant(P>0.05);compared with the raw material of the leaves of the tree,the content of neutral detergent fiber(NDF)and acid detergent fiber(ADF)in all treatment groups after silage was significantly lower(P<0.05),and the content of ADF and The content of ADF and NDF in the experimental group was significantly higher than that in the CK group(P<0.05),and the difference between experimental groups was not significant(P>0.05);the content of soluble carbohydrates(WSC)in all treatment groups was significantly lower compared with that before silage(P<0.05),and the WSC content in all experimental groups was significantly higher than that in the CK group(P<0.05),and the difference in WSC content between experimental groups was not significant(P>0.05).2,inoculation of different sources of lactic acid bacteria improved the fermentation quality of leaf silage in different degrees.The p H values of all treatment groups decreased rapidly in the first 6 d of silage and gradually stabilized with the extension of silage time.The p H values of all experimental groups were significantly lower than those of the CK group(P<0.05),with the lowest in the L2 group and significantly lower than those of the other treatment groups(P<0.05);the lactic acid(LA)contents of all experimental groups were significantly higher than those of the CK group(P<0.05),with the highest LA content in the L2 group and significantly higher than those of the other treatment groups(P<0.05),with significant differences among the treatment groups(P<0.05).The acetic acid(AA)content in the test groups was significantly higher than that in the CK group(P<0.05),with the highest AA content in the L3 group,which was significantly higher than that in the other treatment groups(P<0.05);a small amount of propionic acid(PA)was detected in all treatment groups,with the highest PA content in the CK group,which was significantly higher than that in the other test groups(P<0.05);a small amount of butyric acid(BA)was detected in the CK group,and the No BA was detected in any of the experimental groups;ammoniacal nitrogen(NH3-N/TN)content was significantly lower in all experimental groups than in the CK group(P<0.05),with the lowest NH3-N/TN content in the L2 group,which was significantly lower than in the other treatment groups(P<0.05).4.Effects of inoculating lactic acid bacteria from different sources on microbial diversity during silage fermentation of textured leavesThe aim of this experiment was to investigate the effects of endogenous Lactobacillus L2 strain and exogenous Lactobacillus L3 strain on microorganisms during dynamic fermentation of silage from tree trees.The microbial community structure of the L2 and L3 groups was determined at 4,6,8,30 and 60 d of silage using Miseq high-throughput sequencing technology.The results showed that the microbial community structure of the silage with and without the addition of lactic acid bacteria was changed by Alpha and Beta diversity analysis.The relative abundance of the thick-walled phylum was higher in the L2 group than in the L3 and CK groups from the 4th to the 30 th day of silage;based on the genus level,the dominant genus in the CK group was Klebsiella,the dominant genus in the L2 group was Lactococcus,and the dominant genus in the L3 group was Lactobacillus.Under the same silage time,the relative abundance of the main dominant genera,Lactococcus spp.and Lactobacillus spp.in the L2 and L3 groups were higher than that in the CK group,and the total relative abundance of Klebsiella spp.was lower than that in the CK group.The total relative abundance of Lactococcus spp.and Lactobacillus spp.in the L2 group was higher than that in the L3 group from the 4th to the 30 th day of silage.The results showed that the addition of Lactobacillus spp.to the leaf silage of L.spp.improved the nutritional composition and fermentation quality of the leaf silage of L.spp.and could influence the structure of the microbial community of the silage,increasing the relative abundance of the thick-walled phylum,Lactococcus spp.and Lactobacillus spp.in the Lactobacillus treatment group,reducing the number of harmful microorganisms,and favoring the improvement of the fermentation quality of the leaf silage of L.spp.Among them,the addition of Lactobacillus constructus source was more favorable to the improvement of the silage quality of constructus leaves. |
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