本文選題：乳酸菌 + 篩選　； 參考：《中國農業(yè)大學》2016年博士論文

【摘要】：添加乳酸菌可以增加原料中乳酸菌數量,使青貯盡快進入乳酸菌主導的發(fā)酵階段。然而目前對于可溶性糖較少的牧草,仍然缺乏有效的乳酸菌制劑而不能進行很好地青貯保存。乳酸菌劑在青貯中的應用機理方面的研究還不夠深入,且不同的青貯條件對乳酸菌添加劑的使用效果的影響還需探明。本研究從青貯飼料中分離乳酸菌,篩選生長快產酸高的菌株；研究它們對不同牧草青貯品質,微生物數量,有氧穩(wěn)定性,體外產氣動力學的影響；繼而將篩選到的優(yōu)質菌株在不同的條件下使用,研究原料收獲時期、含水量、WSC含量、青貯密度和貯藏溫度對乳酸菌菌劑添加效果的影響。其主要結果如下：(1)從青貯飼料中篩選得到生長快、產酸高的乳酸菌15株：LB、LPL1、LPL2、LPL3、LCL、 WH、LP1、LP2、LP3、LC1、LC2、PP1、PP2, EF1、EF2。通過常規(guī)生理生化試驗以及16S rDNA序列同源性分析進行鑒定,其中LB屬于短乳桿菌Lactobacillus brevis, LPL1、LPL2、LPL3、LP1、 LP2、LP3屬于植物乳桿菌L. plantarum, LCL、LC1、LC2屬于干酪乳桿菌L. casei, PP1、PP2屬于戊糖片球菌Pediococcus pentosaceus, EF1屬于糞腸球菌Enterococcus faecalis, EF2屬于屎腸球菌E.faecium。(2)單獨添加乳酸菌可提高羊草(Leymus chinnsis)、苜蓿(Medicago sativa)、象草(Pennisetum purpureum)等青貯飼料的發(fā)酵品質,而對天然草地野生牧草青貯發(fā)酵品質的改善效果不明顯。LB、LPL1、LPL2、LPL3、LCL、WH能提高羊草青貯飼料有氧穩(wěn)定性,LB減緩了羊草和賴草(Leymus secalinus)青貯飼料的有氧變敗。LP1和LC1與纖維素酶的混合添加可提高苜蓿和象草青貯飼料的體外消化率。(3)不同條件會影響牧草青貯品質及微生物豐富度a.牧草不同的收獲時期、含水量、青貯密度與貯藏溫度對牧草青貯品質影響很大。羊草在較高青貯密度(600 kg m-3)和較早刈割(抽穗早期)條件下,青貯品質較好,且較早刈割(抽穗早期)的消化性能較好；苜蓿適當晾曬后青貯,青貯品質較好,在20℃,30℃條件下貯藏, Lactobacillus的豐富度最高,.青貯品質更好；b.不同的條件下乳酸菌劑的使用均能改善牧草青貯發(fā)酵品質。LP1和LC1能提高晾曬和未晾曬處理的苜蓿青貯飼料發(fā)酵品質。LP1和LC1提高了兩個時期刈割的羊草青貯飼料的發(fā)酵品質,但對其消化性能沒有改善。LB、LPL1、LPL2、LPL3、LCL、WH在兩個青貯密度條件下均提高了羊草青貯飼料的發(fā)酵品質。在較低青貯密度(500kg m-3)和較晚刈割(抽穗晚期)時,添加乳酸菌也能獲得較好的青貯品質；c.苜蓿青貯飼料在40℃條件下貯藏時,Garciella豐富度較高,丁酸生成量較高；而在20℃和300C條件下,添加LP1和LC1后Lactobacillus比例升高。(4)通過構建工程菌的方式嘗試將木聚糖酶基因xynA導入LP1。將木聚糖酶基因xynA整合到穿梭質粒pMG36e,成功構建重組質粒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，

本文編號：1966309