饲草青贮用乳酸菌的筛选及作用机理

发布时间：2018-06-01 23:58

  本文选题：乳酸菌 + 筛选　； 参考：《中国农业大学》2016年博士论文

【摘要】：添加乳酸菌可以增加原料中乳酸菌数量,使青贮尽快进入乳酸菌主导的发酵阶段。然而目前对于可溶性糖较少的牧草,仍然缺乏有效的乳酸菌制剂而不能进行很好地青贮保存。乳酸菌剂在青贮中的应用机理方面的研究还不够深入,且不同的青贮条件对乳酸菌添加剂的使用效果的影响还需探明。本研究从青贮饲料中分离乳酸菌,筛选生长快产酸高的菌株；研究它们对不同牧草青贮品质,微生物数量,有氧稳定性,体外产气动力学的影响；继而将筛选到的优质菌株在不同的条件下使用,研究原料收获时期、含水量、WSC含量、青贮密度和贮藏温度对乳酸菌菌剂添加效果的影响。其主要结果如下：(1)从青贮饲料中筛选得到生长快、产酸高的乳酸菌15株：LB、LPL1、LPL2、LPL3、LCL、 WH、LP1、LP2、LP3、LC1、LC2、PP1、PP2, EF1、EF2。通过常规生理生化试验以及16S rDNA序列同源性分析进行鉴定,其中LB属于短乳杆菌Lactobacillus brevis, LPL1、LPL2、LPL3、LP1、 LP2、LP3属于植物乳杆菌L. plantarum, LCL、LC1、LC2属于干酪乳杆菌L. casei, PP1、PP2属于戊糖片球菌Pediococcus pentosaceus, EF1属于粪肠球菌Enterococcus faecalis, EF2属于屎肠球菌E.faecium。(2)单独添加乳酸菌可提高羊草(Leymus chinnsis)、苜蓿(Medicago sativa)、象草(Pennisetum purpureum)等青贮饲料的发酵品质,而对天然草地野生牧草青贮发酵品质的改善效果不明显。LB、LPL1、LPL2、LPL3、LCL、WH能提高羊草青贮饲料有氧稳定性,LB减缓了羊草和赖草(Leymus secalinus)青贮饲料的有氧变败。LP1和LC1与纤维素酶的混合添加可提高苜蓿和象草青贮饲料的体外消化率。(3)不同条件会影响牧草青贮品质及微生物丰富度a.牧草不同的收获时期、含水量、青贮密度与贮藏温度对牧草青贮品质影响很大。羊草在较高青贮密度(600 kg m-3)和较早刈割(抽穗早期)条件下,青贮品质较好,且较早刈割(抽穗早期)的消化性能较好；苜蓿适当晾晒后青贮,青贮品质较好,在20℃,30℃条件下贮藏, Lactobacillus的丰富度最高,.青贮品质更好；b.不同的条件下乳酸菌剂的使用均能改善牧草青贮发酵品质。LP1和LC1能提高晾晒和未晾晒处理的苜蓿青贮饲料发酵品质。LP1和LC1提高了两个时期刈割的羊草青贮饲料的发酵品质,但对其消化性能没有改善。LB、LPL1、LPL2、LPL3、LCL、WH在两个青贮密度条件下均提高了羊草青贮饲料的发酵品质。在较低青贮密度(500kg m-3)和较晚刈割(抽穗晚期)时,添加乳酸菌也能获得较好的青贮品质；c.苜蓿青贮饲料在40℃条件下贮藏时,Garciella丰富度较高,丁酸生成量较高；而在20℃和300C条件下,添加LP1和LC1后Lactobacillus比例升高。(4)通过构建工程菌的方式尝试将木聚糖酶基因xynA导入LP1。将木聚糖酶基因xynA整合到穿梭质粒pMG36e,成功构建重组质粒pMG36e-xynA。
[Abstract]:The addition of lactic acid bacteria can increase the number of lactic acid bacteria in raw materials and make the silage enter the fermentation stage dominated by lactic acid bacteria as soon as possible. However, forage with less soluble sugar is still lack of effective lactic acid bacteria preparation and can not be well silage. The study on the application mechanism of lactic acid bacteria in silage is not enough, and the influence of different silage conditions on the effect of lactic acid bacteria additive needs to be explored. In this study, lactic acid bacteria were isolated from silage to screen strains with fast growth and high acid production, and their effects on silage quality, microbial quantity, aerobic stability and gas production kinetics in vitro were studied. The effects of raw material harvest time, water content and WSC content, silage density and storage temperature on the effect of adding lactic acid bacteria were studied. The main results were as follows: (1) 15 strains of lactic acid bacteria with fast growth and high acid production were obtained from silage. By routine physiological and biochemical tests and homology analysis of 16s rDNA sequence, The LB belongs to Lactobacillus brevis, LPL1 to LPL2LPL3LP1, LP2LP3 to Lactobacillus plantarum, LCLLLC1LC2 to Lactobacillus casei, PP1 to Pediococcus pentosaceus, EF1 to Enterococcus faecalis. and EF2 to E. faecium. The fermentation quality of silage, such as Leymus chinnsis, Medicago sativaa and Pennisetum purpureum, etc. But the effect of improving the fermentation quality of wild forage silage in natural grassland was not obvious. LBN LPL1, LPL2, LPL3, LCL-WH could improve aerobic stability of Leymus silage and Leymus secalinus) silage. LP1, LC1 and cellulase were mixed with cellulase in the silage of Leymus chinensis and Leymus secalinus. The in vitro digestibility of alfalfa and elephant grass silage was increased. The water content, silage density and storage temperature have great influence on the quality of forage silage. Under the conditions of higher silage density (600kg m ~ (-3) and early cutting (early heading stage), the silage quality of Leymus chinensis was better, and the digestibility of early cutting (early heading) was better, and the silage quality of alfalfa was better after proper drying. The richness of Lactobacillus was highest when stored at 20 鈩，

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