苜蓿青贮中梭菌多样性及其诱发梭菌发酵的机理研究

发布时间：2018-03-15 04:20

本文选题：梭菌　切入点：多样性　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：苜蓿由于水分含量高、可溶性碳水化合物含量低及缓冲能高等原因,直接青贮极易发生梭菌发酵导致失败。该文旨在研究参与苜蓿青贮发酵的梭菌种类、数量以及不同发酵时期优势梭菌菌群的演替规律,初步阐明梭菌增殖诱发梭菌发酵降低苜蓿青贮品质的机理。主要研究内容及结论如下:1.利用变性梯度凝胶电泳技术研究了 4个品种苜蓿[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)后密封贮藏14、28及56天后的青贮中的梭菌菌群构成。结果表明,Cl.sporgenes在未萎蔫A4青贮梭菌发酵过程中起主导作用,而其他3品种未萎蔫苜蓿青贮中梭菌发酵主要与Cl.perfringens的活动相关。与未萎蔫苜蓿青贮相比,萎蔫处理显著抑制了梭菌发酵,Cl.ghonii及Cl.sartagoforme的活动可能与萎蔫2-h组的苜蓿青贮(水分含量为64.6%-66.8%)中梭菌发酵相关;萎蔫4-h组的苜蓿青贮(水分含量为56.2%-58.4%)中梭菌发酵被完全抑制。2.利用高通量测序技术研究了苜蓿青贮过程中梭菌菌群动态变化。试验设置添加乳酸菌接种剂(105cfu/gFM)及蔗糖(20g/kgFM)的LS组以及未添加的CK组,于发酵后的第0、3、7、14、28 及 56 天取样分析。结果表明,Romboutsia sp.、Clostridium tyrobutyricum、Cl.perfringens、Garciella sp.及Terrisporobacter mayombei是附着在苜蓿表面的主要梭菌。青贮过程中,LS组发酵品质良好,其梭菌菌群构成在青贮前后无显著差异。CK组发生严重梭菌发酵,Cl.perfringens、Garciellasp.及Cl.baratii在发酵前3天快速增殖,随后Cl.perfringens及Cl.baratii成为优势梭菌,14天后Cl.tyrobutyricum在梭菌菌群中占据主导地位。因此推测,Cl.perfringens、G arciella sp.及Cl.baratii启动了 CK组苜蓿青贮过程中梭菌发酵,而Cl.tyrobutyricum主要在梭菌发酵后期起作用。3.利用分离纯培养法研究了苜蓿青贮中优势梭菌的菌群构成及其生理生化特性。根据形态学特征及生理生化试验将分离的65株梭菌划分为5个组,并结合16S rDNA序列分析将其分别鉴定为: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。与其他4种梭菌相比,Cl.perfringens可以利用多种碳源底物并且可以在45℃及50℃的高温条件下生长。因此,有必要进一步研究 Cl.perfringens在梭菌发酵过程中的作用。4.利用高通量测序技术研究了接种Cl.perfringens对苜蓿青贮中梭菌菌群构成的影响。试验设置添加乳酸菌接种剂(105cfu/gFM)及蔗糖(20g/kgFM)的LS组以及未添加的CK组,在发酵后的第14、28及42天分别接种Cl.perfringen(103 cfu/g FM),并分别以调制青贮或接种梭菌为时间原点发酵56天后取样分析。结果表明,接种梭菌对LS组发酵过程无显著影响,而接种梭菌促进了 CK组梭菌发酵。与Clostridium相比,随贮藏时间延长,Garciella在CK组梭菌发酵过程中的作用更大。CK组青贮过程中,第28天接种梭菌组的梭菌发酵程度均比第14天或42天接种组更严重,这可能也与Garciella的活动有关。与Cl.perfringens相比,Garciella sp.及Cl.tyrobutyricum在CK组梭菌菌群中占据优势地位。因此推测,Garciella sp.及Cl.tyrobutyricum 的活动对促进CK组梭菌发酵的作用更大,而Cl.perfringens在促进梭菌发酵过程中的作用仍需进一步研究。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ920.1

【参考文献】