本文關(guān)鍵詞： 腸道抗病菌 細(xì)菌素 菌株分離 發(fā)酵條件優(yōu)化　出處：《江西農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究通過對(duì)健康成年豬腸道內(nèi)生菌群的分離,利用點(diǎn)種法篩選出對(duì)臨床分離的致病性大腸桿菌有明顯抑制作用的腸道活性菌株作為研究對(duì)象,通過排除酸、過氧化氫試驗(yàn)、酶解試驗(yàn)來初步確定活性菌株所產(chǎn)抑菌活性產(chǎn)物為多肽或蛋白類物質(zhì)。通過對(duì)活性菌株盲腸2號(hào)菌菌落形態(tài)、代謝試驗(yàn)及16S r DNA的檢測(cè),鑒定其為枯草芽孢桿菌。通過對(duì)活性菌株盲腸2號(hào)菌生產(chǎn)活性產(chǎn)物時(shí)的接種量、初始p H、培養(yǎng)溫度、發(fā)酵時(shí)間、培養(yǎng)基組成的研究,確定了該菌株生產(chǎn)活性物質(zhì)的最佳條件,為今后進(jìn)一步開發(fā)和利用該活性菌株奠定了基礎(chǔ)。主要內(nèi)容包括以下三個(gè)部分:1.豬腸道內(nèi)生菌群活性菌株的分離。從健康成年豬腸道不同區(qū)段(十二指腸、空腸、回腸、盲腸、結(jié)腸、直腸)的菌群中進(jìn)行腸道內(nèi)生菌株的分離和培養(yǎng),分離得到形態(tài)特征盡可能不同的菌落共計(jì)2317株,以臨床分離的致病性大腸桿菌(Escherichia coli)作為指示菌,點(diǎn)種法對(duì)峙培養(yǎng),將對(duì)指示菌具有明顯抑菌作用的腸道內(nèi)生菌株進(jìn)行分離純化,作為候選活性菌株。利用牛津杯瓊脂擴(kuò)散法對(duì)這些候選活性菌株的發(fā)酵上清液的抑菌活性進(jìn)行檢測(cè),排除產(chǎn)酸、過氧化氫的可能后,再進(jìn)行胰蛋白酶、木瓜蛋白酶的酶解處理,將酶解后發(fā)酵上清抑菌活性明顯降低或完全喪失的菌株推定為產(chǎn)蛋白質(zhì)類抑菌物質(zhì)的活性菌株,留作進(jìn)行后續(xù)研究。2.活性菌株的鑒定。以活性菌株盲腸2號(hào)菌的菌落特征、革蘭氏染色,以及各種代謝反應(yīng)試驗(yàn)結(jié)果為依據(jù),初步確定盲腸2號(hào)菌株為芽孢桿菌(Bacillus)。進(jìn)一步通過對(duì)盲腸2號(hào)菌的16S r DNA測(cè)序結(jié)果:盲腸2號(hào)菌株與枯草芽孢桿菌(Bacillus subtilis)同源性達(dá)到99%以上,鑒定本試驗(yàn)篩選所得盲腸2號(hào)菌為枯草芽孢桿菌。3.盲腸2號(hào)菌株生產(chǎn)活性產(chǎn)物時(shí)的最佳生產(chǎn)條件研究。通過研究不同接種量、初始p H、培養(yǎng)溫度、發(fā)酵時(shí)間、培養(yǎng)基組成對(duì)活性菌株盲腸2號(hào)菌生產(chǎn)活性產(chǎn)物的影響,結(jié)果表明該菌株在利用PCA肉湯,接種量為3%、初始p H為7.0、培養(yǎng)溫度為37℃、發(fā)酵時(shí)間為6h時(shí)生產(chǎn)活性物質(zhì)的抑菌活性最強(qiáng)。
[Abstract]:Through the isolation of intestinal endophytic bacteria from healthy adult pigs, the enteric active strains with obvious inhibitory effect on clinical pathogenic Escherichia coli were screened by point breeding method. The bacteriostatic products of the active strains were identified as polypeptide or protein by enzymatic hydrolysis test. The colony morphology, metabolic test and 16s r DNA of the active strain cecum 2 were detected. Bacillus subtilis was identified as Bacillus subtilis. The optimum conditions for the production of active substances were determined by studying the inoculation amount, initial pH, culture temperature, fermentation time and composition of culture medium of the active strain cecum 2. It lays a foundation for the further development and utilization of this active strain. The main contents include the following three parts: 1. Isolation of the active strains of endophytic bacteria from the intestinal tract of healthy adult pigs (duodenum, jejunum, ileum, cecum, intestinal tract, ileum, cecum, duodenum, jejunum, ileum, cecum). The intestinal endophytic strains were isolated and cultured in colon and rectum. A total of 2317 strains with different morphologic characteristics were isolated. Escherichia coli, which is clinically isolated, was used as an indicator. The enteric endophytic strains with obvious bacteriostatic effect were isolated and purified as candidate active strains. The inhibitory activity of the fermentation supernatants of these candidate active strains was detected by Oxford cup Agar diffusion method, and the acid production was excluded. After the possibility of hydrogen peroxide, trypsin and papain were treated by enzymatic hydrolysis. The strains that had obviously decreased or completely lost the bacteriostatic activity of the supernatant of fermentation after enzymatic hydrolysis were presumed to be active strains producing protein-producing antimicrobial substances. 2. Identification of the active strains. Based on the colony characteristics of the active strain cecum 2, Gram staining, and the results of various metabolic reactions. The cecum 2 strain was identified as Bacillus subtilis. The result of 16s r DNA sequencing showed that the homology of cecum 2 strain and Bacillus subtilis was more than 99%. Cecum 2 was identified as Bacillus subtilis. 3. The optimum production conditions of cecum 2 were studied. Different inoculation amount, initial pH, culture temperature and fermentation time were studied. The effect of the composition of culture medium on the production of active product of Cecum 2 strain showed that the strain was using PCA broth, the inoculation amount was 3%, the initial pH was 7.0, and the culture temperature was 37 鈩，

本文編號(hào)：1496414