Microbiology And Metabonomics Study Of High-quality Alfalfa Silage Controlled By Inoculants And Enzymes

The application of silage alfalfa in ruminants is gradually increasing,but it difficult to make high-quality silage because the high buffer value and the low content of water-soluble carbohydrates in alfalfa.Silage additives affect the fermentation and nutritional quality of silage by affecting the microbial community and their metabolites during silage fermentation and aerobic exposure.Study of bacterial communities in silage is not thorough enough due to the limitation of research technology.The recent development of new-generation DNA sequencing technology facilitates the detailed analysis of bacterial diversity in silage.Inoculants,especially lactic acid bacteria,can use the soluble carbohydrates in silage to produce organic acids in fermentation,reduc ing p H and inhibiting the growth of spoilage bacteria in silage,and thus effectively maintain the nutrient composition of silage.Different enzyme preparations can decompose the corresponding substrates in the silage to produce soluble carbohydrates,and also promote the propagation of lactic acid bacteria.In this study,alfalfa(Medicago sativa)was selected as the research subject and explored by combining the methods and techniques of nutrition,high-throughput sequencing,metabolomics,bioinformatics and morphology.Firstly,the correlations between bacterial diversity and fermentation quality of alfalfa silage treated with different inoculants,or enzymes,or the combination of enzyme and inoculants.Then the correlation between microbial community diversity and fermentation quality of alfalfa silage treated with inoculants during aerobic exposure was explored.Finally,the succession,morphology and metabolomics of alfalfa silage treated with enzymes and inoculants were investigated.(1)Correlation between bacterial diversity and fermentation quality of alfalfa silage treated with different inoculantsThe bacterial diversity of alfalfa silage treated with Lactobacillus plantarum(LP),Bacillus subtilis(BS),or LP + BS(LPBS)and its correlation with fermentation quality were studied on an Illumina Miseq sequencing platform.The p H of silage significantly decreased,and the contents of dry matter and lactic acid significantly rose after any treatment,indicating the inoculant treatments improved the silage quality.Microbiota analysis showed the bacterial diversity of silage was affected by inoculant treatment.The dominant bacterial communities in the untreated silage were Garciella(30.3%)and Enterococcus(24.8%).Lactobacillus was dominant in LP silage and LPBS silage with abundance of 98.2 % and97.2%,respectively.Lactobacillus also dominated in BS silage with abundance of 73.8%.Lactobacillus was significantly correlated negatively with p H of any treated silage(LP,BS and LPBS),but positively with lactic acid content.Garciella and Enterococcus were significantly positively correlated with both p H and propionic acid content in the untreated silage.LP silage and LPBS silage were of higher quality,which was related to Lactobacillus.(2)Correlation between bacterial diversity and fermentation quality of alfalfa silage treated with enzymes or the enzyme/inoculant combinationThe bacterial communities of alfalfa silage treated with cellulase + ?-galactosidase(CEGA),cellulase + L.plantarum(CELP),or ?-galactosidase + L.plantarum(GALP),and their relationships with fermentation quality were analyzed.The fermentation quality of silage was significantly improved after all treatments,including a decreased in p H,and ammonia nitrogen content and an increase in lactic acid content(P< 0.05).Microbiota analysis showed enzyme treatment changed the composition and diversity of bacterial communities in silage.Lactobacillus was the dominant bacterial community in both CELP silage and GALP silage with the abundance of 97.88% and 96.74%,respectively.Lactobacillus and Pediococcus were dominant in CEGA silage with the abundance of 49.64%and 35.31%,respectively.The abundance of Garciella was the highest in the untreated silage(30.21%),while the abundance of Lactobacillus was only 12.11%.Correlation analysis showed Lactobacillus was positively correlated with the lactic acid content in both CELP silage and GALP silage,while Garciella and Enterococcus were positively correlated with the p H,propionic acid and butyric acid content s in the untreated silage.Pediococcus and Weissella were positively correlated with acetic acid content in CEGA silage.CELP w as the best treatment,and its silage quality was related to Lactobacillus.(3)Correlation between microbial community diversity and fermentation quality of alfalfa silage treated with inoculants during aerobic exposureThe microbial diversity of alfalfa silage treated with Lactobacillus paracasei(LC)during three sampling periods of aerobic exposure was studied,including 0 d(PO0),7 d(PO7)and 14 d(PO14).The p H of silage increased and the lactic acid content decreased after aerobic exposure.LC silage showed significantly less p H and higher lactic acid levels during aerobic exposure than the untreated silage(P< 0.05).Microbial analysis showed the bacterial communities were affected by aerobic exposure.Lactobacillus,Enterobacteria and Enterococcus were the dominant bacteria in the untreated silage,and the abundance of Enterobacteria and Enterococcus decreased gradually during aerobic exposure.Lactobacillus was dominant in the LC silage with abundance gradually decreasing during aerobic exposure.The abundance of Acetobacter increased significantly in both the untreated silage and the treated silage in PO14.Correlation analysis showed Lactobacillus was correlated positively with lactic acid content,but negatively with p H.Enterobacteria,Enterococcus,Weissella,Cedecea and Sporolactobacillus were correlated significantly and negatively with lactic acid content,but positively with p H(P< 0.05).Acetobacter was negatively and significantly correlated with acetic acid content(P< 0.05).Fungal communities are also affected by aerobic exposure.At the phylum level,Ascomycota was dominant in the untreated silage with abundance increasing from 67.57% on day 0 to 99.51% on day 14.Basidiomycota abundance decreased from 32.43% on day 0 to0.49% on day 14.In the LC silage,Ascomycetes abundance was maintained between 94.79%and 99.93%.At the genus level,unclassified?Eurotiales was the dominant species in the untreated silage with abundance increasing from 30.83% on day 0 to 90.52% on day 14.In the LC silage,the abundance of unidentified?Saccharomycetes increased from 44.55% on day0 to 93.99% at 14 d.Correlation analysis did not find any correlation between fungus and silage quality.L.paracasei treatment improved alfalfa silage quality both in ensiling and during aerobic exposure,and the silage quality was related to Lactobacillus.(4)Succession,morphology and metabolomics of alfalfa silage treated with enzyme and inoculantsAlfalfa was treated with Lactobacillus casei(LC)and Cellulase(CE)separately.Samples were taken for analysis at ensiling of 0 d,7 d,56 d and 3 d of aerobic exposure.Compared with the untreated silage,the enzyme and inoculant treatment decreased the p H,and increased lactic acid yield and quality of silage significantly(P< 0.05).Totally 1095 operational taxonomic units(OTUs)belonging to 27 phyla and 511 genera were identified by Illumina sequencing.Totally of 764 OUTs from 422 genera and 21 phyla were obtained from fresh alfalfa,and Sphingobium was the most abundant.The bacterial diversity decreased after silage treatment.Lactobacillus developed slowly in the untreated silage and its abundance was only 37.3% on day 7,75.10% on day 56,and 42.02% after aerobic exposure.Lactobacillus developed rapidly in the CE silage,reaching 93.19% on day7 and 83.93% on day 56,and decreasing to 79.69% after aerobic exposure.In the LC silage,the abundance of Lactobacillus reached 98.37% on day 7 and 94.7% on day 56,and was still91.70% after aerobic exposure.Enterococcus also accounted for certain abundance in the untreated silage.Scanning electron microscopy showed the wax layer was intact in the fresh alfalfa,but was decomposed almost completely in the untreated silage and partially in both CE silage and LC silage.The main bacterium attached to the untreated leaf surface was cocci.Both cocci and bacilli were found on the surface of CE leaves,and mainly bacilli appeared on the leaf surface of LC silage.Metabonomics results showed 196 metabolites in the samples after 3 days of aerobic exposure,including 112 metabolites that showed significant differences among groups(P<0.05).The metabolites contained the most diverse and up to 52 types of organic acids.The silage was found with 19,22,18 and 8 types of sugars,alcohols,esters,and amino acids respectively.Various organic acids,alcohols,aldehydes and ketones have antibacterial activity and positively affect aerobic stability.Correlation analysis showed vari ous metabolites were significantly associated with Lactobacillus,such as lactic acid,2-hydroxybutyric acid,valine,isoleucine,hydrocinnamic acid,3-(4-hydroxyphenyl)propionic acid,benzoic acid,succinic acid,2-deoxyerythritol,N-acetyl-L-leucine,acetyl alcohol,2,5-dihydroxybenzaldehyde,D-arabinitol,threonic acid,and acetylsalicylic acid(P< 0.05).In addition,hydrocinnamic acid,3-(4-hydroxyphenyl)propionic acid,benzoic acid,succinic acid,2-deoxyerthritol,N-acetyl-L-leucine,acetol,2,5-dihydroxybenzaldehyde,and D-arabinol were significantly associated with Lactococcus and Enterococcus(P< 0.05).In conclusion,inoculants and enzyme can improve the quality and lactic acid yield of alfalfa silage,and reduce p H.Both inoculants and enzyme can increase the abundance of Lactobacillus in silage,but the increasing abilities of LAB inoculants surpassed those of enzyme and non-LAB inoculants.After short-term aerobic exposure,the metabolites of alfalfa silage treated with inoculants and with enzymes differed,and most metabolites were correlated with LAB species.Inoculant treatment can also im prove the stability of bacterial community and metabolites of aerobic exposed alfalfa silage,and thus improves the quality of aerobic exposed alfalfa silage.Cellulase + L.plantarum,Lactobacillus casei and Lactobacillus paracasei can better improve silage quality than other inoculants and enzyme preparations,and are recommended as the additives for silage.