| Enzyme preparations are receiving increasing attention in improving silage quality,but they are mainly targeted at enhancing the nutrient content of silage and improving fermentation quality.It is not clear about how enzymes affect the complex microbial processes during fermentation and their related mechanisms.In the present experiment,silage of maize and alfalfa was mixed with different concentrations of cellulase(0.04 g/kg,0.05 g/kg,0.06 g/kg)and hemicellulase(0.3 g/kg,0.4 g/kg,0.5g/kg)in the ratio of 6:4,and the dynamic changes in the nutritional quality of silage at different time periods(0d,7d,14 d,30d,45d)were investigated.The dynamic changes of silage nutritional quality at different time periods(0d,7d,14 d,30d and 45d)were investigated,and combined with 16 S r RNA high-throughput sequencing of microbial diversity to elucidate how enzymes affect silage quality through the regulation of microorganisms in the silage fermentation process.The main findings were as follows:1.Sensory assessment results of the silages of each treatment: after 45 days of silage,the p H values of the silages of the seven treatment groups ranged from 3.68 to3.72 after opening the cans,none of them appeared moldy,all the stem and leaf structures remained intact,the texture was loose,and the color was yellowish green;the sensory scores were divided into two grades,the control group,0.04 g/kg and 0.05g/kg cellulase treatment group treatments were good,and the remaining four The other four treatments were excellent.2.The nutrient content of each treatment changed during the silage process:during the silage process,the WSC content,NDF and ADF of each treatment showed a significant decreasing trend;the DM content decreased in the early stage and increased after 30 d;the CP content decreased in the first 30 d and then tended to be stable.The CP loss rate was the lowest in the group treated with 0.06 g/kg cellulase and 0.5 g/kg hemicellulase after 45 d of silage(P?0.05);the DM content was the highest in the group treated with 0.5 g/kg hemicellulase(P?0.05),and the NDF and ADF contents were the lowest in the group treated with 0.06 g/kg cellulase(P?0.05).The subordinate function analysis of sensory and nutritional quality showed that the groups treated with 0.5 g/kg hemicellulase and 0.06 g/kg cellulase performed better.3.Analysis of fermentation quality of 0.5 g/kg hemicellulase and 0.06 g/kg cellulase treated groups and CK: The p H value of each group showed a decreasing trend with the extension of silage time,and the p H value of each treatment was less than 3.8 after 45 d of silage.there was no significant difference(P?0.05)in the content of ammonia nitrogen between the three experimental groups after 45 d of silage.the content of LA and AA showed a gradual increase with the extension of silage time.The LA and AA contents increased gradually with the increase of silage time,and the LA content of the group treated with 0.06 g/kg cellulase at 45 d of silage was higher than the other groups(P?0.05).The group treated with 0.06 g/kg cellulase showed the least change in AA content during the whole process.PA and BA were not detected in all groups in all periods.4.High-throughput sequencing of microorganisms in the 0.5 g/kg hemicellulase and 0.06 g/kg cellulase treatment groups and CK:(1)High-throughput sequencing using 16 Sr RNA amplification technology,665 families,1526 genera and 1955 species of bacteria and 354 families,853 genera and 1545 species of fungi were detected in the three treatment groups;(2)The ACE index and Chao index of bacterial diversity in each treatment group increased with the extension of silage fermentation time.The ACE and Chao indices of bacterial diversity in each treatment group showed an increasing trend with the extension of silage fermentation time,and the Simpson index showed a decreasing trend followed by an increasing trend(P<0.05),except that the Simpson index of bacteria in the CK group was significantly lower than the other two groups at 3 d(P<0.05);the ACE and Chao indices of fungal diversity in each treatment group fluctuated up and down with the extension of silage fermentation time,and the Simpson The ACE index and Chao index of fungal diversity in each treatment group fluctuated with the extension of silage fermentation time,and the Simpson index and Shannon index showed a trend of decreasing and then increasing(P<0.05).(3)With the extension of silage time,different additives led to dramatic changes in bacterial flora,the largest difference in the number of unique bacteria OUT in the group with 0.06 g/kg cellulase added,increased by 945 after 45 d.The group with 0.5 g/kg hemicellulase added increased by 827.the smallest difference in the CK group increased by 764;the group with 0.06 g/kg cellulase added increased by 764 after 45 d.The fungal OTU,decreased by 123,added 0.5 g/kg hemicellulase group and increased by 127 after 45 d.The analysis of bacterial composition showed that the dominant bacterial group before silage in each treatment was Proteobacteria,which changed to Lactobacillus after silage;the addition of 0.06g/kg cellulase could alleviate the decrease in the abundance of Lactobacillus during silage;the dominant fungal group was Firmicutes.The dominant groups of fungi were Basidiomycota and Kurtzmaniella.(5)Correlation analysis showed that the addition of additives caused a significant positive effect of Lactobacillus spp.and an unclassified cyanobacterium on WSC content and eliminated the significant effect of the bacterial community on LA and AA content.The effect of Sphingomonas spp.on DM content became insignificant after the addition of cellulase,and the addition of hemicellulase caused a significant effect of an unclassified Escherichia coli on cellulose content.The addition of hemicellulase reduced the effect of Saccharomyces cerevisiae on CP and NDF and changed the negative correlation on p H;the effect of Streptomyces on NDF was reduced.The addition of hemicellulase caused a significant effect of a variety of fungi on the content of nutrients and effectively inhibited the growth of undesirable microorganisms,thus significantly improving the quality of silage. |
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