| Native grass is the most important material basis in grassland livestock production.With the high-quality development of animal husbandry,silage is increasingly favoured by farmers.However,it is often difficult to ensilage successfully due to its low moisture and water soluble carbohydrate content,as well as the low number of epiphytic lactic acid bacteria.In order to ensure the fermentation quality of native grass silage,native grasses from the meadow steppe,typical steppe and desert steppe of Xilingol grassland in Inner Mongolia were studied in this paper.The nutritional quality,fermentation quality,microbial abundance,bacterial community structure and metabolic characteristics of native grass silage were analyzed during the ensiling and aerobic exposure processes based on the research methods and techniques of nutrition,microbiomics,metabolomics and bioinformatics.The aim of the study was to reveal the microecosystem fermentation mechanism of the native grass silage,screen and evaluate excellent lactic acid bacteria specialized for native grass silage,and provide important theoretical and technical support for the resarch of fermentation mechanism of native grass silage.The main research findings were as follows:(1)There were some differences in the nutritional quality and microecological environment of the native grass from three steppe types.The native grass of meadow steppe had the highest water soluble carbohydrate content(1.95%)(P>0.05),and the number of epiphytic lactic acid bacteria(4.78 lg cfu/g)was significantly higher than in the desert steppe(P<0.05).The native grass of meadow steppe having a richer bacterial community,with Pantoea agglomerans having the highest abundance in meadow steppe and typical steppe,and Rosenbergiella nectarea having the highest abundance in native grass of desert steppe.A total of 340 differential metabolites were screened from native grass of three steppes,there were mainly categorised as amino acids,sugars,alcohols,esters and ketones and aldehydes.The pathways that differential metabolites were enriched to were mainly cysteine and methionine metabolism,purine metabolism,α-linolenic acid metabolism,and the biosynthesis of unsaturated fatty acids.(2)The ensiling time and steppe types had significant effects(P<0.05)on the nutritional quality,fermentation quality and microbial population of native grass silage.The trends of dynamic changes in the nutritional quality,fermentation quality and microbial population of native grass silage in the three steppe types were basically consistent.Ensiling can retain the maximum nutritional quality of native grass and reduce the number of harmful microorganisms.In microecological fermentation system of native grass silage,the bacterial community completed the succession from Gram-negative to Gram-positive bacteria with a decrease in diversity.Among them,Levilactobacillus brevis and Lentilactobacillus buchneri dominated fermentation from 15 d of ensiling in meadow steppe,Limosilactobacillus fermentum dominated fermentation from 30 d of ensiling in typical steppe,and Lactiplantibacillus brevis and Lactiplantibacillus plantarum dominated fermentation from 15 d of ensiling in desert steppe.(3)Aerobic exposure time and steppe types significantly(P<0.05)affected the nutritional quality,fermentation quality and microbial population of native grass silage.The dry matter,crude protein,water soluble carbohydrates,lactic acid and acetic acid contents of native grass silage in three steppes decreased significantly(P < 0.05),increasing the number of harmful microorganisms,such as yeasts,aerobic bacteria,Escherichia coli and molds,as the number of days of aerobic exposure increased.Exposure time was the main factor affecting the silage quality of native grass silage,and steppe types also had a significant effect on water soluble carbohydrates,crude protein,neutral detergent fiber content,p H value,and the number of lactic acid bacteria and yeasts during aerobic exposure of ensiling.The aerobic stability of native grass silage in meadow steppe,typical steppe and desert steppe were 150 h,110 h and 145 h,respectively.(4)A total of 67 strains of lactic acid bacteria were screened from native grass raw materials and silages of meadow steppe,typical steppe and desert steppe,and were identified as belonging to 10 species by 16 S r RNA gene sequences.Through screening studies on physiological and biochemical characteristics,tolerance,acid production,and growth rate,there were determined that Limosilactobacillus fermentum(MB17)、Leuconostoc citreum(MB42)、Latilactobacillus graminis(MB56)、Lactiplantibacillus plantarum(MB83)and Pediococcus acidilactici(MB104)were suitable candidates for native grass silage fermentation.(5)Five strains of preferred lactic acid bacteria and two strains of commercial lactic acid bacteria affected the silage quality and microbial population of native grass to varying degrees.Each of the lactic acid bacteria additives significantly reduced the p H value and ammonia nitrogen content of native grass silage(P<0.05),significantly increased the number of lactic acid bacteria and reduced the number of yeasts and general aerobic bacteria(P<0.05).Among them,MB17,MB56 and MB83 screened in this study had better effect on silage quality enhancement of native grass,and could be used as special lactic acid bacteria additives for silage preparation of native grass.On this basis bacterial community and metabolic characterisation of native grass silage in the MB17,MB56 and MB83 treatment groups.The dominant bacterial species in the MB17 treatment were Limosilactobacillus fermentum and Lentilactobacillus buchneri.The dominant strains in the MB56 and MB83 treatment groups were Lactiplantibacillus plantarum,Lentilactobacillus buchneri,and Levilactobacillus brevis.Amino acid metabolism and carbohydrate metabolism played decisive role in native grass silage,and the most metabolite-enriched pathways in the MB17,MB56 and MB83 treatment groups were the arginine and proline metabolism pathways,the metabolism pathways of glycine,serine and threonine,and the cysteine and methionine metabolism pathways. |
