Screening And Characterizing Of Ferulic Acid Esterase-producing Lactic Acid Bacteria And Their Application In Alfalfa Silage

Ferulic acid esterase(FAE)is a subclass of the carbohydrate esterase subfamily,which is able to hydrolyze the ester bond between ferulic acid and polysaccharides in the cell walls of a plant and breaks down its network structures,releasing ferulic acid and their dimers.FAE is widely used in the release of ferulic acid,biological synthesis,paper industry,production of fuel ethanol,and animal feed production.In this paper,we screened and characterized the enzymatic and physicochemical properties offerulic acid esterase-producing lactic acid bacteria,and studied the their application in alfalfa silage as well.1.Five strains of lactic acid bacteria that produce feruloyl esterasewere screened by assaying the transparent zone on the selective ager plate with ethyl ferulate as the main carbon source.The enzyme activity was determined by High Performance Liquid Chromatography.By analyzing 16 S rRNA,phylogenetic analysis,and Species-specific PCR,the five strains were identified to be Lactobacillus plantarum A1,Lactobacillus brevis A3,and Pediococcus acidilacticis A2,A4,and A5.2.The characteristics of ferulic acid esterase produced by the five strains were analyzed,and the results shown that when methyl ferulate was the substrate,the optimum pH of FAE was 6.4,the optimum temperature was 37 °C,and the strains was stable in the pH range of 5.0 to 7.0and at the temperature range from 25 to 50 °C.The physiological and biochenmical characteristics,sugar utilization ability of the five lactic acid bacteria were analyzed,and it was shown that all the strains were found to be tolerant to the growth environment.These strains could grow between the temperatures of 10 to 45°C,pH 3.5 to 8.5,and concentration of NaCl from 3% to 8%.Moreover,the five strains could ferment most of the sugar,having an extensive ability on fermenting carbohydrates.3.The Lactobacillus plantarumA1(LP),Lactobacillus brevis A3(LBr),commercial inoculants Lactobacillus plantarum(ALP),and Cellulase(CE)were inoculated to alfalfa at ensiling,and ensiled 7 d,30 d,and 60 d at room temperature(~25°C).The additive treatments were reduced pH levels and increased lactic acid concentrations(P<0.001),but it did not inhibit the degradation of protein.The use of cellulase alone did not improve the nutritional value of the silage and did not affect neutral detergent fiber and acid detergent fiber.Further,silage produced with Lactobacillus plantarum A1(LP)had lesserneutral detergent fiber and acid detergent fiber concentrations than the control.In conclusion,application of ferulic acid esterase-producing lactobacilli to alfalfa silage improved the fermentation quality of alfalfa silage,and the use of Lactobacillus plantarumA1 reduced the fiber content,indicating that the ferulic acid esterase-producing Lactobacillus plantarum A1 used in silage not only could promote fermentation,improve silage quality,and reduce the fiber content,but also could improve the feed value compared with the traditional lactic acid bacteria.This subsequently solves the problem of low sugar content,low lactic acid bacteria,low fiber degradation rate,and low success rate of alfalfa silage.