| Silage is an important feed resource that makes up over 50% of dairy diets fed to dairy cows.It is an indispensable type of forage for the high-quality development of modern herbivorous animal husbandry.It is also a breakthrough and main starting point for ensuring the quality and safety of livestock products and the effective implementation of “grain-to-forage”.Effectively improving the technical level of standardized production of silage in my country can provide important technical support for accelerating the development of modern grass husbandry in China.The development and utilization of silage lactic acid bacteria preparations play an important role in the production of high-quality silage and the development of the forage industry,and it is one of the important guarantee measures to promote the safe and efficient development of the forage industry.The main function of the commonly used silage lactic acid bacteria preparation is to improve the fermentation quality of silage and prevent mildew.At present,the development of silage lactic acid bacteria preparations that can not only improve the fermentation quality of silage,but also have the function of degrading fibers has always been a hot spot in the field of silage lactic acid bacteria research.Based on the current international situation of silage fermentation regulation and a large number of previous studies,this thesis screened out a strain of ferulic acid esterase-producing Lactobacillus plantarum A1 that can effectively change the lignocellulose structure in the silage fermentation process.And through the high lignocellulose material silage and enzymatic hydrolysis and saccharification experiments,the mechanism of changing the structure of lignocellulose was explained.Later,in vitro rumen fermentation test and livestock digestion and metabolism test proved its effect on forage digestibility and livestock health as a silage additive.The main results obtained in this study are as follows:1.The application research of ferulic acid esterase-producing Lactobacillus plantarum A1 in corn stalk silage shows that adding Acremonium cellulase(AC),inoculating with Lactobacillus plantarum A1(Lp),or adding AC and Lp in corn stalk silage at room temperature(～25℃)can reduce silage pH,increase lactic acid content to improve the fermentation quality of silage.Lactobacillus plantarum A1 alone does not have high degradation performance on structural carbohydrates during ensiling,but the combined use of Lactobacillus plantarum A1 and Acremonium cellulase has a better degradation effect on lignocellulose.After 60 days of ensiling,the a NDF,ADF,hemicellulose,and cellulose in the AC+Lp treatment increased by 9.38%,17.2%,8.24%,and 18.8% respectively compared with the control group.However,in the enzymatic saccharification experiment,the enzymatic digestibility of the Lp treatment was significantly higher than that of the control,AC and AC+Lp treatments.The enzymatic digestibility of the Lp treatment was 23.3%,26.7%,43.3% higher than that of the control,AC and AC+Lp treatments,respectively.However,the excellent feature of Lactobacillus plantarum A1 only worked at 25°C rather than 40°C.This provides guidance for the future application of Lactobacillus plantarum A1 in silage and the pretreatment of biofuel production.2.The application research of ferulic acid esterase-producing Lactobacillus plantarum A1 in different dry matter Pennisetum sinese silage shows that silage treated with Acremonium cellulase(AC),Lactobacillus plantarum A1(Lp)or their combination can preserve the forage well pass lowering the pH of silage and promote the degradation of lignocellulose as compared with the control.In the low dry matter(L-DM)silage,the AC treatment had a better degrading effect of lignocellulose.After60 days of silage,a NDF,ADF,hemicellulose,and cellulose could increase by 10.4%,7.19%,17.1%,8.31%,respectively,compared with the control group.The AC+Lp treatment had the highest degradation rate,which was 11.5%,7.64%,19.4%,8.31%higher than the control group’s a NDF,ADF,hemicellulose,and cellulose degradation rates,respectively.The digestibility of enzymatic saccharification was the highest in the AC+Lp treatment,which was significantly higher than that in the AC and Lp treatment groups,and the control group was the lowest.The cellulose conversion efficiency of AC+Lp,AC and Lp treatments were 60.0%,45.0%,and 40.0% higher than those of the control group,respectively.In the high dry matter(L-DM)silage,the Lp treatment had a better degrading effect of lignocellulose.After 60 days of silage,a NDF,ADF,hemicellulose,and cellulose could increase 5.38%,5.36%,6.48%,and6.17%,respectively,compared with the control group.Similarly,the AC+Lp treatment had the highest degradation rate,which was 7.87%,7.84%,9.26%,10.21% higher than the control group’s a NDF,ADF,hemicellulose,and cellulose degradation rates,respectively.The digestibility of enzymatic saccharification was the highest in the Lp treatment,which was significantly higher than that in the AC and AC+Lp treatments,while the control group was the lowest.The cellulose conversion efficiency of Lp,AC and AC+Lp treatments were 317%,283%,and 250% higher than those of the control group,respectively.The inoculation of Lactobacillus plantarum A1 and the addition of Acremonium cellulase in the silage both reduced the crystallinity of the lignocellulose of silage,thereby increasing the accessibility of cellulase to polysaccharides,and further improving L-DM or H-DM cellulose conversion efficiency of silage lignocellulose.In L-DM silage,the use of Acremonium cellulase can effectively degrade the lignocellulose of P.sinese silage and improve the degree of enzymatic hydrolysis and saccharification of the silage.However,when P.sinese is ensiled at the H-DM level,it is recommended to use Lactobacillus plantarum A1 alone or in combination with Acremonium cellulase.3.The effect of ferulic acid esterase-producing Lactobacillus plantarum A1 on the fermentation quality and antioxidant properties of alfalfa silage shows that the inoculation of Lactobacillus plantarum A1(Lp A1)during alfalfa ensiling can significantly improve the fermentation quality,reduce the pH value of the silage,and increase its lactic acid concentration.After 90 d of ensiling,the lowest DM loss and non-protein N were observed in the Lp A1 inoculated silage.At the same time,inoculation of Lp A1 also decreased the neutral detergent fiber,acid detergent fiber,and acid detergent lignin concentrations in the later period of ensiled alfalfa,and increased ferulic acid concentration during all ensiling duration.The highest ferulic acid concentration was detected in Lp A1 treated silage after 30 d(P < 0.05).In addition,the total antioxidant capacity and glutathione peroxidase activity in the Lp A1 and Lp 24-7 inoculated silages were higher than that in the CON and Lp MTD/1silages from 30 d to 90 d of ensiling.However,lower lipoxygenase activity was found in Lp A1 and Lp 24-7 inoculated silages during the whole fermentation process.Both Lp A1 and Lp 24-7 improved the total fatty acid concentration and the proportion of polyunsaturated fatty acids in total fatty acid of ensiled alfalfa when compared with the CON and Lp MTD/1 silages after 90 d of fermentation(P < 0.05).Therefore,inoculation of ferulic acid esterase-producing Lp A1 or antioxidant strain Lp 24-7 not only improved the fermentation quality and chemical composition of ensiled alfalfa but also enhanced the antioxidant status of the silage.4.The results of in vitro fermentation experiments show that the inoculation of ferulic acid esterase-producing lactic acid bacteria in alfalfa silage can improve the in vitro dry matter digestibility and total gas production,but has no effect on methane production.Inoculation of ferulic acid esterase-producing lactic acid bacteria in alfalfa silage can significantly promote rumen fermentation and increase the concentration of total volatile fatty acids and ammonia nitrogen in rumen fluid,especially the concentrations of acetic acid,propionic acid,butyric acid and branched chain fatty acids.In addition,inoculation of ferulic acid esterase-producing lactic acid bacteria in alfalfa silage has little effect on the microbial diversity of the rumen fluid,but it can significantly increase the number of cellulolytic bacteria and carbohydrate utilization bacteria,such as Rumencoccus albus,Rumenococcus flavefaciens and Prevotella.5.Dairy goat digestion and metabolism experiments show that alfalfa wrapped silage inoculation of Lp A1 has better fermentation quality than the commonly used strain Lp MTD/1,and can significantly improve total antioxidant capacity of silage,and the enzymatic activity of superoxide dismutase,and glutathione.Compared with the Lp MTD/1 treatment,alfalfa wrapped silage inoculation with Lp A1 can significantly increase the digestibility of dry matter,organic matter and crude protein in dairy goats.In addition,inoculation of Lp A1 in silage can significantly promote the rumen fermentation of dairy goats and increase the concentration of total volatile fatty acids,acetic acid,propionic acid and isobutyric acid.The diet of the Lp A1 treatment for dairy goats can significantly increase their serum total antioxidant capacity and antioxidant enzyme activity,but has little effect on the antioxidant performance of milk serum.The serum immunoglobulin A concentration of dairy goats in the Lp A1 treatment group was significantly higher than that of the Lp MTD/1 treatment,but the concentrations of TNF-α,IL-2 and IL-6 were significantly lower than those of the Lp MTD/1 treatment.In addition,compared with the Lp MTD/1 treatment,inoculation of Lp A1 in the silage has a smaller effect on the milk production of dairy goats,but has a greater impact on the milk composition,which can significantly increase milk fat,milk protein,total solids and urea content.For the first time,this study comprehensively explained the mechanism of ferulic acid esterase-producing lactic acid bacteria to degrade the structure of lignocellulose,and proved that inoculation of ferulic acid esterase in silage can significantly improve the digestibility of forage by in vivo and in vitro experiments.In addition,inoculation of ferulic acid esterase-producing lactic acid bacteria in silage has a positive effect on the health of livestock.This provides important technical support for the application of ferulic acid esterase-producing lactic acid bacteria in silage. |
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