兰州大学机构库 >生命科学学院
产阿魏酸酯酶乳酸菌的筛选、酶学特性及其在苜蓿青贮中的应用研究
Alternative TitleScreening and characterizing of ferulic acid esterase-producing lactic acid bacteria and their application in alfalfa silage
荆佩欣
Thesis Advisor郭旭生
2017-06-11
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword乳酸菌 阿魏酸酯酶 酶学特性 苜蓿青贮
Abstract

阿魏酸酯酶是碳水化合物酯酶亚家族的一种,能够水解植物细胞壁中阿魏酸与多糖间的酯键,打破它们的网状结构,释放出阿魏酸或阿魏酸二聚体。阿魏酸酯酶在阿魏酸的释放、生物合成、造纸工业、生产乙醇及动物饲料生产等诸多方面应用广泛。本论文主要从产阿魏酸酯酶乳酸菌的筛选、鉴定、酶学特性、菌株理化特性以及在苜蓿青贮中的应用等几方面进行了研究,结果如下。1. 采用以阿魏酸乙酯为唯一碳源的平板透明圈法筛选到5株产阿魏酸酯酶的乳酸菌,并用高效液相色谱法测定其酶活。同时通过16S rRNA基因鉴定、系统进化分析及Species-specific PCR得知,5株乳酸菌分别为植物乳杆菌(Lactobacillus plantarum)A1,短乳杆菌(Lactobacillus brevis)A3和3株乳酸片球菌(Pediococcus acidilactici)A2,A4,A5。2. 分别对5株乳酸菌的阿魏酸酯酶粗酶液进行了酶学特性的分析,结果表明5株菌阿魏酸酯酶粗酶液的最适反应pH为6.4,最适反应温度为37 °C,且在pH为5.0~7.0,温度为25~50 °C的条件时,具有较好的稳定性。同时,对5株乳酸菌菌株的理化特性及糖发酵进行了研究,发现5株乳酸菌对生长环境具有较强的耐受性,在温度为10~45 °C、pH 3.5~8.5及3%~8% 浓度的NaCl条件下均可生长,而且,能利用大部分的糖源,对糖源的利用能力比较广泛。3. 将筛选得到的产阿魏酸酯酶酶活较高植物乳杆菌A1(LP)和短乳杆菌A3(LBr),以及商品化植物乳杆菌(ALP),纤维素酶(CE)作为青贮添加剂添加到苜蓿青贮中,室温(约25 °C)下青贮7 d,30 d,60 d。发现所有添加剂处理组均显著降低了pH,乳酸含量增加(P<0.001)。但对青贮饲料中的蛋白降解未起到抑制作用。单独添加纤维素酶时,未能改善青贮饲料营养价值,且对纤维含量也没有影响。此外,只有在单独添加产阿魏酸酯酶的植物乳杆菌时降低了青贮饲料的纤维含量。综上所述,将产阿魏酸酯酶的乳杆菌应用到到苜蓿青贮中时,改善了苜蓿青贮品质,且添加植物乳杆菌降低了青贮的纤维含量。说明产阿魏酸酯酶的植物乳杆菌在青贮发酵的过程中降解了饲料纤维。与传统乳酸菌相比,不仅可以促进发酵,改善青贮品质,而且可以降低纤维,提高饲料的饲养价值。同时,为解决天然牧草低的含糖量、附着乳酸菌少、纤维降解率低、青贮成功率低等问题提供一定的技术支持,有效提高天然牧草的储存和利用。

Other Abstract

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 16S 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.

URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/221639
Collection生命科学学院
Recommended Citation
GB/T 7714
荆佩欣. 产阿魏酸酯酶乳酸菌的筛选、酶学特性及其在苜蓿青贮中的应用研究[D]. 兰州. 兰州大学,2017.
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