本文選題：梭菌　切入點：多樣性　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：苜蓿由于水分含量高、可溶性碳水化合物含量低及緩沖能高等原因,直接青貯極易發(fā)生梭菌發(fā)酵導(dǎo)致失敗。該文旨在研究參與苜蓿青貯發(fā)酵的梭菌種類、數(shù)量以及不同發(fā)酵時期優(yōu)勢梭菌菌群的演替規(guī)律,初步闡明梭菌增殖誘發(fā)梭菌發(fā)酵降低苜蓿青貯品質(zhì)的機理。主要研究內(nèi)容及結(jié)論如下:1.利用變性梯度凝膠電泳技術(shù)研究了 4個品種苜蓿[Sanditi(A1)、AC Caribou(A2)、WL319HQ(A3)及4030(A4)]未萎蔫(0-h)或萎蔫2h及4h(2-h、4-h)后密封貯藏14、28及56天后的青貯中的梭菌菌群構(gòu)成。結(jié)果表明,Cl.sporgenes在未萎蔫A4青貯梭菌發(fā)酵過程中起主導(dǎo)作用,而其他3品種未萎蔫苜蓿青貯中梭菌發(fā)酵主要與Cl.perfringens的活動相關(guān)。與未萎蔫苜蓿青貯相比,萎蔫處理顯著抑制了梭菌發(fā)酵,Cl.ghonii及Cl.sartagoforme的活動可能與萎蔫2-h組的苜蓿青貯(水分含量為64.6%-66.8%)中梭菌發(fā)酵相關(guān);萎蔫4-h組的苜蓿青貯(水分含量為56.2%-58.4%)中梭菌發(fā)酵被完全抑制。2.利用高通量測序技術(shù)研究了苜蓿青貯過程中梭菌菌群動態(tài)變化。試驗設(shè)置添加乳酸菌接種劑(105cfu/gFM)及蔗糖(20g/kgFM)的LS組以及未添加的CK組,于發(fā)酵后的第0、3、7、14、28 及 56 天取樣分析。結(jié)果表明,Romboutsia sp.、Clostridium tyrobutyricum、Cl.perfringens、Garciella sp.及Terrisporobacter mayombei是附著在苜蓿表面的主要梭菌。青貯過程中,LS組發(fā)酵品質(zhì)良好,其梭菌菌群構(gòu)成在青貯前后無顯著差異。CK組發(fā)生嚴重梭菌發(fā)酵,Cl.perfringens、Garciellasp.及Cl.baratii在發(fā)酵前3天快速增殖,隨后Cl.perfringens及Cl.baratii成為優(yōu)勢梭菌,14天后Cl.tyrobutyricum在梭菌菌群中占據(jù)主導(dǎo)地位。因此推測,Cl.perfringens、G arciella sp.及Cl.baratii啟動了 CK組苜蓿青貯過程中梭菌發(fā)酵,而Cl.tyrobutyricum主要在梭菌發(fā)酵后期起作用。3.利用分離純培養(yǎng)法研究了苜蓿青貯中優(yōu)勢梭菌的菌群構(gòu)成及其生理生化特性。根據(jù)形態(tài)學(xué)特征及生理生化試驗將分離的65株梭菌劃分為5個組,并結(jié)合16S rDNA序列分析將其分別鑒定為:Cl.tyrobutyricum(24.6%)、Cl.sordellii(7.7%)、Cl.sporogenes(18.5%)、Cl.subterminale(7.7%)及Cl.perfringens(41.5%),其中Cl.pefringens的含量最高。與其他4種梭菌相比,Cl.perfringens可以利用多種碳源底物并且可以在45℃及50℃的高溫條件下生長。因此,有必要進一步研究 Cl.perfringens在梭菌發(fā)酵過程中的作用。4.利用高通量測序技術(shù)研究了接種Cl.perfringens對苜蓿青貯中梭菌菌群構(gòu)成的影響。試驗設(shè)置添加乳酸菌接種劑(105cfu/gFM)及蔗糖(20g/kgFM)的LS組以及未添加的CK組,在發(fā)酵后的第14、28及42天分別接種Cl.perfringen(103 cfu/g FM),并分別以調(diào)制青貯或接種梭菌為時間原點發(fā)酵56天后取樣分析。結(jié)果表明,接種梭菌對LS組發(fā)酵過程無顯著影響,而接種梭菌促進了 CK組梭菌發(fā)酵。與Clostridium相比,隨貯藏時間延長,Garciella在CK組梭菌發(fā)酵過程中的作用更大。CK組青貯過程中,第28天接種梭菌組的梭菌發(fā)酵程度均比第14天或42天接種組更嚴重,這可能也與Garciella的活動有關(guān)。與Cl.perfringens相比,Garciella sp.及Cl.tyrobutyricum在CK組梭菌菌群中占據(jù)優(yōu)勢地位。因此推測,Garciella sp.及Cl.tyrobutyricum 的活動對促進CK組梭菌發(fā)酵的作用更大,而Cl.perfringens在促進梭菌發(fā)酵過程中的作用仍需進一步研究。
[Abstract]:Alfalfa with high water content, soluble carbohydrate content and low buffer can cause higher, prone to direct silage fermentation of Clostridium cause failure. This paper aims to study the involvement of alfalfa silage fermentation of Clostridium species, quantity and advantages of different fermentation periods of Clostridium bacteria succession law, clarify the proliferation induced by Clostridium Clostridium fermentation mechanism to decrease the quality of alfalfa silage the main research contents and conclusions are as follows: 1. using denaturing gradient gel electrophoresis of 4 varieties of Alfalfa ([Sanditi A1), AC Caribou (A2), WL319HQ (A3) and 4030 (A4)] (0-h) or without wilting wilting of 2H and 4H (2-h, 4-h) Clostridium bacteria after sealed storage 14,28 and after 56 days of silage constitute. The results showed that Cl.sporgenes did not play a leading role in the A4 during the fermentation of Clostridium wilting silage, while the other 3 varieties without wilting alfalfa silage fermentation and Clostridium Cl.perfringens live Dynamic correlation. Compared with non wilted alfalfa silage, wilt was significantly inhibited by Cl.ghonii and Clostridium fermentation, the activities of Cl.sartagoforme and 2-h groups may be wilted alfalfa silage (64.6%-66.8% water content) in Clostridium fermentation; wilt 4-h group of alfalfa silage (56.2%-58.4% water content) in Clostridium fermentation was completely inhibited.2. of Alfalfa in the process of silage Clostridium flora changes using high-throughput sequencing technology. The test set of lactic acid bacteria inoculation agent (105cfu/gFM) and sucrose (20g/kgFM) of LS group and CK group were not added, analysis after fermentation at 0,3,7,14,28 and 56 days of sampling. The results showed that Romboutsia sp., Clostridium tyrobutyricum, Cl.perfringens, Garciella, sp. Terrisporobacter and mayombei are attached to the main surface of the alfalfa silage. Clostridium LS fermentation process, the quality is good, the bacterial composition in Clostridium before silage No significant difference between the.CK group of serious Clostridium fermentation, Cl.perfringens, Garciellasp. and Cl.baratii in the fermentation of 3 days before the rapid proliferation of Cl.perfringens and Cl.baratii, then become the dominant Clostridium, 14 days after the Cl.tyrobutyricum occupies a dominant position in Clostridium bacteria. So, Cl.perfringens G, arciella sp. and Cl.baratii launched the CK group in the process of Clostridium alfalfa silage fermentation, while Cl.tyrobutyricum mainly in late.3. clostridial fermentation using the separation of Clostridium dominant alfalfa silage in the pure culture method of flora composition and physiological and biochemical characteristics. According to the morphological characteristics and physiological and biochemical tests, 65 strains of Clostridium were divided into 5 groups, combined with 16S rDNA sequence analysis, which were identified as: Cl.tyrobutyricum (24.6%), Cl.sordellii (7.7%), Cl.sporogenes (18.5%), Cl.subterminale (7.7%) and Cl.perfringens (41.5%), Cl.pefr Ingens was the highest. Compared with other 4 kinds of Clostridium, Cl.perfringens can use a variety of carbon substrates and can be grown in a high temperature condition of 45 DEG and 50 DEG C. Therefore, it is necessary to study the effect of Cl.perfringens on Clostridium bacteria inoculation of alfalfa silage of high-throughput sequencing technology using.4. for further study of Cl.perfringens in C. during the fermentation process. The test set of lactic acid bacteria inoculation agent (105cfu/gFM) and sucrose (20g/kgFM) of LS group and CK group were added, after fermentation at 14,28 and 42 days were inoculated with Cl.perfringen (103 cfu/g FM), and modulated silage or inoculated Clostridium as the time origin after 56 days fermentation sample analysis. The results showed that the inoculation of Clostridium had no significant effect on group LS fermentation process, and promote the CK group inoculated Clostridium Clostridium fermentation. Compared with Clostridium, with the extension of storage time, the Garciella in the CK group shuttle Fermentation process a larger role in.CK group during ensiling, Clostridium fermentation degree twenty-eighth days inoculated Clostridium group were fourteenth days or 42 days of inoculation was more serious. It may also be related with Garciella. Compared with Cl.perfringens, Garciella, sp. and Cl.tyrobutyricum occupy the dominant position in the CK group of shuttle bacteria. It was suggested that Garciella sp. and Cl.tyrobutyricum CK group activities to promote the clostridial fermentation more important role in promoting Cl.perfringens in fermentation process of Clostridium effect still need further study.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ920.1

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