Mechanism Of Lactiplantibacillus Plantarum A345 Improving Fermentation Quality Of Alfalfa Silage

Alfalfa(Medicago sativa)is a leguminous forage with high nutritional value and an important component of diets of dairy cows.North China,where Henan Province is located,is the main producing area of animal husbandry in China.In Henan Province,it is urgent to speed up the development of alfalfa industry and improve the technical level of alfalfa harvesting and processing.To improve the fermentation quality of alfalfa silage in Yellow River beach in Henan Province,experiments were carried out as following:(1)To evaluate the effects of Lactiplantibacillus plantarum A345 as additive in alfalfa silage on industry-scale,fermentation properties and microbial community compositions in alfalfa silage(800 kg per bag)inoculated with commercial silage additive and L.plantarum A345 were compared.Alfalfa was wilted in the field to a dry matter of 39.70%FM.At 60 days,inoculation of silage additives significantly decreased pH,increased lactic acid accumulation,and inhibited ammoniacal nitrogen formation in silage.Analysis of microbial communities indicated that inoculation of L.plantarum A345 significantly decreased the relative abundance of Pediococcus and Weissella,and increased the relative abundance of Lactiplantibacillus in bacterial community in silage.Meanwhile,it also decreased the relative abundance of Saccharomycetales in fungi community in silage.Inoculation of L.plantarum A345 effectively promoted pH decline and inhibition of ammoniacal nitrogen accumulation and yeast growth comparing with the commercial additive,and improved fermentation quality of alfalfa silage.L.plantarum A345,thus,is more suitable for application as an additive in alfalfa silage production on industry-scale.(2)To evaluate the effects of L.plantarum A345 as additive in high moisture alfalfa silage,fermentation properties and microbial community compositions in alfalfa silage with a dry matter of 26.64%FM inoculated with commercial L.plantarum inoculant and L.plantarum A345 were compared.The L.plantarum inoculants significantly increased relative abundance of Lactobacillus,and promoted lactic acid accumulation for both periods.At 90 d,silage in the non-inoculated group exhibited a high pH,a high concentration of ammoniacal nitrogen,and a severe dry matter loss.The inoculations significantly inhibited the growth of Clostridia and reduced ammoniacal nitrogen concentration in silage.Inoculation of L.plantarum A345 effectively increased the relative abundance of Lactobacillus,and promoted pH decline and lactic acid accumulation comparing with the commercial L.plantarum inoculant.Besides,it perfomed stronger inhibition of spoilage microorganisms,especially Clostridium in silage comparing with the commercial L.plantarum inoculant.Thus,inoculation with L.plantarum A345 improved the fermentation quality of alfalfa silage by regulation of bacterial community,and further delayed spoilage of high moisture alfalfa silage.While the sole inoculation of L.plantarum A345 failed to achieve optimum fermentation quality of high moisture alfalfa silage.(3)To illustrate the dynamics of microbial communities during alfalfa ensiling more comprehensively,bacterial composition and its interaction with fermentation properties of silage indicated by absolute quantification 16S-seq(AQS)and conventional relative quantification 16S-seq(RQS)were compared.Results indicated that variation in total bacterial DNA amounts across different treatments and ensiling periods was illustrated by AQS.AQS indicated higher bacterial richness indices and closer correlations of these indices with fermentation properties than RQS via spearman’s correlation analyses,as well as more taxa with significance on bacterial abundance via lefse analyses.Overall,application of AQS illustrated more comprehensive dynamics of bacterial communities during the ensiling proces.(4)To explore the mechanism of moisture of alfalfa influencing the ensiling process,the dynamics of the fermentation products,residual non-structural carbohydrates,and bacterial communities in non-wilted and wilted alfalfa silage were compared.The non-wilted alfalfa had a dry matter of 23.71%FM.The wilted alfalfa had a dry matter of 37.15%FM.For the wilted silage,the bacterial community,pH value,and ammonia nitrogen concentration remained stable in the silage at 30 days.Inoculation of L.plantarum A345 accelerated lactic acid fermentation and altered the predominant genus in the wilted silage as compared with the non-inoculated group.For the non-wilted group,fast consumption of water-soluble carbohydrates was observed at 10 days in the non-inoculated silage along with rapid growth of undesirable Hafnia.Inoculation of L.plantarum A345 inhibited growth of Hafnia at 10 days in the nonwilted silage.Clostridia fermentation occurred in the non-wilted silage at 90 days,as indicated by apparent abundance of genera belonging to Clostridia,formation of butyric acid,and an increased pH.Meanwhile,galactose content was largely consumed at this ensiling period.Inoculation of L.plantarum A345 inhibited growth of Garciella and butyric acid accumulation in the non-wilted silage at 90 days as compared with the non-wilted silage without inoculation,but had little effect on the growth of Clostridium sensu stricto.Overall,the high moisture content of the non-wilted alfalfa silage led to rapid consumption of water-soluble carbohydrates and growth of harmful microorganisms at the early stage of ensiling.Lack of carbohydrates as fermentation substrates might be a main cause for insufficient lactic acid fermentation in high moisture alfalfa silage.Dynamics of bacterial communities and curves of dynamics of different non-structural carbohydrates during ensiling varied among the treatments.This indicated variations in the utilization efficiency of microbes on different nonstructural carbohydrates.(5)To evaluate the effects of different fermentation substrates along with L.plantarum A345 inoculant on fermentation quality of high moisture alfalfa silage,direct-cut alfalfa with a dry matter of 21.63%FM was ensiled with the following treatments:solely inoculated with L.plantarum A345;and inoculated with L.plantarum A345 along with galactose,lactose,glucose,and molasses.All the four sugar sources promoted pH decline,and lactose and glucose significantly increased lactic acid accumulation in silage comparing with the sole inoculation of L.plantarum A345 at 7 days.After ensiled for 45 days,silage inoculated with L.plantarum A345 along with lactose performed lower pH value comparing with other treatments.Overall,inoculation with L.plantarum A345 along with sugar sources enhanced lactic acid fermentation at early stage of ensiling in high moisture alfalfa silage.Inoculation with L.plantarum A345 along with lactose effectively improved the fermentation quality of high moisture alfalfa silage,which contributed to preservation of silage.