發(fā)布時間：2018-05-14 00:16

  本文選題：發(fā)酵乳酸菌1.2133 + 理化特性�。� 參考：《安徽農業(yè)大學》2017年碩士論文

【摘要】：畜禽抗生素的濫用對畜禽產品造成了藥物殘留和耐藥性菌株的污染等問題,嚴重危害了動物性食品的質量與安全,造成了嚴重的社會公共衛(wèi)生和食品安全。動物微生態(tài)制劑是綠色抗生素替代品之一,具有廣泛的應用價值。乳酸菌是研制動物微生態(tài)制劑的典型菌種,對動物腸道內環(huán)境微生態(tài)的穩(wěn)定具有調節(jié)作用,對宿主腸道疾病具有防治作用。雞白痢沙門氏菌(Salmonella pullorum)是危害養(yǎng)雞業(yè)最嚴重的腸道細菌病之一,是重要的公共衛(wèi)生問題。防治S.pullorum對雞的感染,對保障家禽健康養(yǎng)殖和動物性食品質量與安全具有重要的意義。在乳酸菌屬中,乳酸桿菌是最主要的一類,其中包括干酪乳桿菌(Lactobacillus casei)、發(fā)酵乳桿菌(Lactobacillus fermentum)、嗜酸乳桿菌(Lactobacillus acidophilus)等。L.fermentum天然存在腸道具有很強的胃腸道環(huán)境耐受力等優(yōu)點,有關其在預防和治療S.pullorum感染的報道較少。因此,本研究旨在體外篩選出能抑制S.pullorum生長的乳酸菌株,以此為基礎,研究其體內外對S.pullorum的抗感染作用,進一步闡述乳酸菌對雞抗病原菌感染的保護性作用研究。具體研究內容和結果如下:1.乳酸菌理化特性及抑菌活性比較在本實驗室乳酸菌篩選的基礎上,選取L.casei1.2435、L.fermentum1.2133、L.acidophilus1.1854、S.thermophillus 1.2471,4株乳酸菌進行抑菌特性比較分析。通過耐酸性和膽鹽耐受實驗、細胞黏附實驗、疏水性實驗、抑菌實驗進行乳酸菌理化特性比較。實驗結果表明,4株乳酸菌在pH值2.5的培養(yǎng)基中培養(yǎng)2h后,L.fermentum1.2133的存活率達到68%,顯著高于其他3株菌。在膽鹽濃度為0.3%的培養(yǎng)基中培養(yǎng)2h后,L.fermentum1.2133存活率大于60%。表面疏水性實驗結果表明,各菌株間比較差異顯著,L.fermentum 1.2133具有較高的表面疏水性;各菌株對目標菌株均具有凝聚能力,其中L.fermentum1.2133自聚合能力最強。抑菌實驗結果表明,4株乳酸桿菌對病原菌E.coli AE17、E.coli O78以及S.pullorum CVCC533均具有一定的抑菌能力,且存在菌株差異性。L.fermentum1.2133對S.pullorum CVCC533有顯著的抑菌能力,L.acidophilus1.1854對E.coli AE17抑菌活性顯著,L.casei1.2435對E.coli O78抑菌能力顯著,S.thermophillus1.2471抑菌活性較弱。2.L.fermentum1.2133拮抗S.pullorum CVCC533體外抑菌作用觀察采用熒光顯微鏡對L.fermentum1.2133發(fā)酵上清作用于S.pullorum CVCC533生物被膜中細菌的生存能力進行觀察,在L.fermentum1.2133發(fā)酵上清液與S.pullorum CVCC533共培養(yǎng)24h后,顯微鏡中觀察到實驗組中S.pullorum CVCC533的生物被膜中S.pullorum CVCC533菌體大部分呈現(xiàn)紅色熒光,只有少量點狀的綠色熒光;對照組視野中基本為綠色熒光,僅有少量紅色熒光。在體外對L.fermentum1.2133與S.pullorum CVCC533進行競爭性共培實驗的結果表明,在0-18h,L.fermentum1.2133對S.pullorum CVCC533的生長抑制隨著時間的推移逐漸增強,S.pullorum的數(shù)量逐漸減少,到18h后,趨于穩(wěn)定。L.fermentum1.2133對S.pullorum CVCC533生長具有拮抗作用,且不同的初始菌液濃度形成的競爭性效果存在差異,菌液濃度10:1的競爭性優(yōu)于1:1。3.L.fermentum1.2133對感染S.pullorum CVCC533的雞腸道菌群和組織病理學觀察將120只1日齡的羅曼肉雞隨機分為4個組,分別為對照組(第I組)、1日齡開始灌喂L.fermentum1.2133(2.5×108 cfu/chick)組(第II組)、1日齡開始灌喂L.fermentum1.2133至14日齡攻毒S.pullorum CVCC533(5×108 cfu/chick)的預處理組(第III組)以及第14日齡直接感染S.pullorum CVCC533組(第IV組),分別進行腸道細菌計數(shù)和病理切片觀察。結果表明,雞感染后24h,回腸和盲腸細菌數(shù)均發(fā)生變化,第II組乳酸菌數(shù)量顯著高于第III組、第IV組,而沙門氏菌數(shù)量明顯有所減少,好氧菌總數(shù)增多。第II組雞的十二指腸、空腸、回腸腸狀態(tài)正常,腸絨毛完整,無明顯病理變化較第I組雞小腸形態(tài)發(fā)育更完整;第III組雞十二指可見腸絨毛固有層出現(xiàn)輕微腸炎性細胞侵潤;空腸絨毛中心軸有部分紅細胞,回腸絨毛表現(xiàn)局部損傷。第IV組十二指腸絨毛斷裂,絨毛自溶現(xiàn)象明顯,空腸絨毛中心軸紅細胞累積,回腸腸絨毛上皮的杯狀細胞被溶解,絨毛自溶現(xiàn)象明顯,第III組與第IV組相比較,雞小腸各腸段的炎癥和出血點等癥狀減輕。4.L.fermentum1.2133對感染S.pullorum CVCC533的雞腸道AvBD2 mRNA轉錄水平的影響本實驗采用RT-PCR方法,測定了L.fermentum1.2133對感染S.pullorum CVCC533的雞腸道AvBD2 mRNA的轉錄水平。在雞感染72 h內(0 h,12 h,24 h,36、48h和72h)采雞小腸各段組織。結果表明,AvBD2在小腸各段中均有表達。在整個時間段內,第II組AvBD2 mRNA的轉錄水平在雞小腸內的轉錄水平基本呈穩(wěn)定趨勢且轉錄水平顯著高于第I組。第III組和第IV組小腸段AvBD2 mRNA的轉錄水平與第II組差異顯著;第III組在24h后,AvBD2 mRNA的轉錄水平基本呈波動性變化趨勢降低,第IV組呈顯著性下降,都顯著低于第I組轉錄水平,實驗結果表明,L.fermentum1.2133對雞腸道防御素AvBD2的表達具有調控作用。上述研究表明,L.fermentum1.2133對雞腸道內環(huán)境具有很好耐受能力,且其發(fā)酵上清液對S.pullorum CVCC533生物被膜的中S.pullorum CVCC533有顯著的抑菌作用;能穩(wěn)定定植于腸道,對雞腸道菌群有調節(jié)作用,同時還能促進雞小腸內β-防御素2的表達量,在抗S.pullorum CVCC533感染過程中發(fā)揮著重要的作用。
[Abstract]:The abuse of livestock and poultry antibiotics caused the drug residues and the contamination of drug resistant strains, seriously endangering the quality and safety of animal food, causing serious social public health and food safety. Animal microecological preparation is one of the substitute of green antibiotics and has extensive application value. Lactic acid bacteria is the development of the animal products. The typical strains of animal microecological agents regulate the stability of the microecological environment in the intestinal tract of animals and have the control effect on the intestinal diseases of the host. Salmonella pullorum is one of the most serious intestinal bacterial diseases endangering the poultry industry, and it is an important public health problem. The prevention and control of S.pullorum infection to chickens. It is of great significance to protect the health of poultry and the quality and safety of animal food. Lactobacillus is the most important category in the genus Lactobacillus, including Lactobacillus casei (Lactobacillus casei), Lactobacillus fermentum, Lactobacillus acidophilus (Lactobacillus acidophilus) and other natural intestinal tract. With the advantages of strong tolerance to the gastrointestinal environment and so on, there are few reports on the prevention and treatment of S.pullorum infection. Therefore, this study aims to screen out the lactic acid strains that can inhibit the growth of S.pullorum in vitro. On the basis of this study, the anti infection effect of S.pullorum in vitro and in vitro and in vivo is studied, and the resistance of lactic acid bacteria to the chicken is further elaborated. Study on the protective effect of dyeing. The specific contents and results are as follows: 1. comparison of physical and chemical properties and bacteriostasis activity of lactic acid bacteria on the basis of lactic acid bacteria screening in our laboratory, the bacteriostasis characteristics of L.casei1.2435, L.fermentum1.2133, L.acidophilus1.1854 and S.thermophillus 1.2471,4 strains were compared and analyzed. Acid resistance and bile salt were used. Tolerance test, cell adhesion experiment, hydrophobic experiment and bacteriostasis experiment were used to compare physical and chemical properties of lactic acid bacteria. The experimental results showed that after 4 strains of lactic acid bacteria were cultured in the medium of pH value 2.5, the survival rate of L.fermentum1.2133 reached 68%, which was significantly higher than that of other 3 strains. After the culture of 2h in the medium with the concentration of bile salt concentration 0.3%, L.fermentum1.2133 survived. The results of the hydrophobicity greater than the 60%. surface showed that there was a significant difference among the strains, and the L.fermentum 1.2133 had a high surface hydrophobicity, and all the strains had the ability to condense to the target strains, and the self polymerization ability of L.fermentum1.2133 was the strongest. The results of bacteriostasis experiment showed that 4 strains of lactobacillus were E.coli AE17, E.coli O78. And S.pullorum CVCC533 all had certain bacteriostasis ability, and the existence of strain differential.L.fermentum1.2133 had significant inhibitory ability to S.pullorum CVCC533, L.acidophilus1.1854 had significant antibacterial activity to E.coli AE17, L.casei1.2435 was significant to E.coli O78, and the inhibitory activity of S.thermophillus1.2471 was weak. The bacteriostasis effect of anti S.pullorum CVCC533 in vitro was observed by fluorescence microscopy on the viability of bacteria in S.pullorum CVCC533 biofilm by L.fermentum1.2133 fermentation supernatant. After L.fermentum1.2133 fermentation supernatant and S.pullorum CVCC533 were co cultured 24h, the S.pullorum CVCC533 in the experimental group was observed in the micromirror. Most of the S.pullorum CVCC533 mycelium in the biofilm showed red fluorescence, only a small number of spot green fluorescence; the control group was basically green fluorescence and only a small amount of red fluorescence. The results of competitive co culture experiment on L.fermentum1.2133 and S.pullorum CVCC533 in vitro showed that in 0-18h, L.fermentum1.2133 to S.pullor. The growth inhibition of UM CVCC533 gradually increased with time, and the number of S.pullorum gradually decreased. After 18h, a stable.L.fermentum1.2133 had an antagonistic effect on S.pullorum CVCC533 growth, and the competitive effects of different initial bacteria concentration were different, and the competitiveness of the liquid concentration 10:1 was better than 1:1.3.L.fermentum1.21. 33 S.pullorum CVCC533 infected chicken intestinal tract flora and histopathological observation of 120 1 day old Roman broilers were randomly divided into 4 groups, respectively, the control group (group I), 1 days of age to start feeding L.fermentum1.2133 (2.5 x 108 cfu/chick) group (group II), 1 days of age to start feeding L.fermentum1.2133 to 14 days old S.pullorum CVCC533 (5 * 108. Cfu/chick) pretreated group (group III) and fourteenth day old direct infection S.pullorum CVCC533 group (group IV), the intestinal bacterial count and pathological section were observed respectively. The results showed that the number of bacteria in the ileum and the cecum were all changed after the chicken infection, and the number of lactic acid bacteria in group II was significantly higher than that of the group III, group IV, and the number of Salmonella was obvious. The number of aerobic bacteria increased. The duodenum, jejunum, ileum and intestinal villi in group II were normal and the intestinal villi were intact. No obvious pathological changes were found in group I. The twelve fingers of group III showed slight enteritis invasion of the lamina propria; the central axis of the jejunum had some red cells and ileum. In group IV, the duodenal villi was broken, the villus autolysis was obvious, the central axis of the jejunum villi was accumulated, the goblet cells in the intestinal villi epithelium were dissolved and the villus autolysis was obvious. Group III was compared with the group IV, and the symptoms of the intestinal segments of the small intestine of the chicken reduced the sense of.4.L.fermentum1.2133 to the feeling. The transcriptional level of AvBD2 mRNA in chicken intestinal tract infected with S.pullorum CVCC533 was influenced by RT-PCR method, and the transcriptional level of AvBD2 mRNA of chicken intestinal tract infected with S.pullorum CVCC533 was measured by L.fermentum1.2133. In chicken infection 72 h (0 h, 12, 24, and so), it was found that all segments of the small intestine of chicken were found in all segments of the small intestine. In the whole time period, the transcriptional level of AvBD2 mRNA in group II was basically stable in the small intestine of the chicken and the transcriptional level was significantly higher than that in the I group. The transcriptional level of AvBD2 mRNA in group III and IV group was significantly different from that in the group II; in the III group, the transcriptional level of AvBD2 AvBD2 was basically fluctuant after 24h. The potential of group IV decreased significantly and was significantly lower than that of group I. The results showed that L.fermentum1.2133 had a regulatory effect on the expression of chicken intestinal defensin AvBD2. The above study showed that L.fermentum1.2133 was well tolerated in the environment of chicken intestinal tract, and the fermentation supernatant had a biofilm of S.pullorum CVCC533 biofilm. S.pullorum CVCC533 has significant bacteriostasis, stable colonization in the intestinal tract, regulating the intestinal flora of Chicken Intestines, and also promoting the expression of beta defensin 2 in the small intestine of chicken, and plays an important role in the anti S.pullorum CVCC533 infection process.

【學位授予單位】：安徽農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.31

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