本文選題：人腸道菌群 + 連續(xù)培養(yǎng)��； 參考：《華中農(nóng)業(yè)大學(xué)》2008年碩士論文

【摘要】： 殘留在動(dòng)物可食性組織中的抗菌藥物被人體攝入后,可能對(duì)人體腸道菌群產(chǎn)生諸多不良影響,如選擇出耐藥的腸道菌群或使腸道細(xì)菌耐藥性增強(qiáng),破壞腸道菌群的屏障作用,改變腸道菌群代謝活性等,從而對(duì)人類健康構(gòu)成巨大威脅。在國(guó)際上,抗菌藥殘留對(duì)人體腸道菌群影響的安全性評(píng)價(jià)研究日漸成為熱點(diǎn)。但國(guó)內(nèi)由于缺乏被國(guó)際組織普遍認(rèn)可的評(píng)價(jià)模型,該領(lǐng)域的研究工作進(jìn)展緩慢。本研究利用細(xì)菌連續(xù)培養(yǎng)的恒化器原理,通過對(duì)稀釋率和轉(zhuǎn)入連續(xù)培養(yǎng)時(shí)間兩方面條件的摸索和優(yōu)化,建立了人腸道微生態(tài)恒化器模擬系統(tǒng)。該模擬系統(tǒng)是國(guó)際組織推薦的兩大類模型之一,其建立將會(huì)促進(jìn)國(guó)內(nèi)外該領(lǐng)域的研究,對(duì)保障人類食品和醫(yī)學(xué)安全具有極其重要的意義。 本研究選用擬桿菌—膽汁—七葉苷瓊脂培養(yǎng)基、BBL瓊脂培養(yǎng)基、伊紅美蘭瓊脂培養(yǎng)基和腸球菌選擇性培養(yǎng)基,分別分離計(jì)數(shù)人糞便中四種優(yōu)勢(shì)代表菌(脆弱擬桿菌、雙歧桿菌、大腸桿菌和腸球菌)。建立糞便中三種主要短鏈脂肪酸(SCFA,即乙酸、丙酸和丁酸)濃度測(cè)定的氣相色譜方法。將50mL(20%)的新鮮糞便懸液接種于發(fā)酵罐中,厭氧條件下分批培養(yǎng),定時(shí)取樣,采用平板稀釋計(jì)數(shù)法記錄培養(yǎng)液中四種優(yōu)勢(shì)代表菌的數(shù)目,繪制其分批培養(yǎng)的菌體生長(zhǎng)曲線,計(jì)算對(duì)數(shù)生長(zhǎng)期。在此基礎(chǔ)上,以培養(yǎng)液中四種優(yōu)勢(shì)代表菌數(shù)目和三種主要的短鏈脂肪酸濃度為監(jiān)測(cè)指標(biāo),優(yōu)化連續(xù)培養(yǎng)工藝,包括稀釋率(0.05h~(-1)、0.07h~(-1)和0.09h~(-1))和轉(zhuǎn)入連續(xù)培養(yǎng)的時(shí)間(第5h、第7h和第9h),建立人腸道微生態(tài)恒化器模擬系統(tǒng)。在優(yōu)化后的培養(yǎng)條件下進(jìn)行為期30 d的試驗(yàn),進(jìn)一步評(píng)價(jià)所建立的模擬系統(tǒng)的穩(wěn)定性和可靠性。 分批培養(yǎng)顯示,四種優(yōu)勢(shì)菌的對(duì)數(shù)生長(zhǎng)期不盡相同,但都包含了第3-8h。在0.07h~(-1)的條件下,系統(tǒng)在連續(xù)培養(yǎng)的前14d達(dá)到了穩(wěn)態(tài),因此0.07h~(-1)為相對(duì)較合適的稀釋率。第7h轉(zhuǎn)入連續(xù)培養(yǎng)時(shí),系統(tǒng)較其他兩個(gè)條件更早達(dá)到了穩(wěn)態(tài),且穩(wěn)態(tài)時(shí)系統(tǒng)中的菌群數(shù)量和SCFA濃度也最高,因此第7h為相對(duì)較合適的轉(zhuǎn)入連續(xù)培養(yǎng)的時(shí)間。30d試驗(yàn)顯示,在第9-30d的系統(tǒng)中,四種代表菌的數(shù)量和三種SCFA的濃度變化差異均不顯著,系統(tǒng)培養(yǎng)條件(如溫度、攪拌速度、pH值等)的穩(wěn)定性也較好。模擬系統(tǒng)在達(dá)到穩(wěn)態(tài)后,其培養(yǎng)體系中的四種優(yōu)勢(shì)菌數(shù)量和三種SCFA濃度與接種前的糞便相比略有下降,但均在報(bào)道的健康人正常的生理參數(shù)范圍內(nèi)。 上述試驗(yàn)結(jié)果表明,本研究通過對(duì)稀釋率和轉(zhuǎn)入連續(xù)培養(yǎng)時(shí)間等條件的摸索和優(yōu)化,成功建立了人腸道微生態(tài)恒化器模擬系統(tǒng),系統(tǒng)具有較好的穩(wěn)定性和可靠性,能夠模擬健康人的腸道微生態(tài)環(huán)境。本研究首次從菌群數(shù)量和代謝物濃度兩個(gè)角度較全面地對(duì)恒化器模擬系統(tǒng)的穩(wěn)定性、可靠性進(jìn)行了評(píng)價(jià),填補(bǔ)了國(guó)內(nèi)研究的空白。本研究還解決了目前國(guó)內(nèi)外建立同類模擬系統(tǒng)時(shí)存在的攪拌不均勻、液位不穩(wěn)定、操作復(fù)雜而導(dǎo)致模擬系統(tǒng)相對(duì)不夠穩(wěn)定等問題。 該恒化器模擬系統(tǒng)的建立可以為抗菌藥殘留對(duì)人腸道菌群影響的安全性評(píng)價(jià)研究構(gòu)建平臺(tái),也可為新藥、功能食品等的安全評(píng)價(jià)研究及藥物代謝研究提供更科學(xué)的方法和途徑,對(duì)保障人類食品和醫(yī)學(xué)安全、促進(jìn)相關(guān)學(xué)科發(fā)展具有極其重要的意義。
[Abstract]:The antimicrobial agents remaining in animal edible tissues may have many adverse effects on the intestinal flora of the human body, such as the selection of drug resistant intestinal flora or the enhancement of antimicrobial resistance of intestinal bacteria, the barrier effect of intestinal flora, and the change of intestinal microflora metabolism, so as to pose a huge threat to human health. At the international level, the research on the safety assessment of the effects of antimicrobial residues on the intestinal flora is becoming a hot topic. However, the research work in this field is slow due to the lack of an evaluation model universally recognized by international organizations. This study uses the principle of continuous culture of the chemostat of bacteria, through two parties to the dilution rate and the time to the continuous culture. The simulation system of human intestinal microecological chemostat is established. This simulation system is one of the two major models recommended by international organizations. The establishment of this simulation system will promote the research in this field at home and abroad. It is of great significance to ensure the safety of human food and medicine.
In this study, bacteriobacterium - bile - seven glucoside agar medium, BBL agar medium, ironorchid agar medium and enterococci selective medium were used to separate four dominant representative bacteria (fragile bacillus, Bifidobacterium, Escherichia coli and Enterococcus) in human feces, and three kinds of main short chain fatty acids (SCFA, b) in feces were established. A gas chromatographic method for the determination of the concentration of acid, propionic acid and butyric acid. The 50mL (20%) fresh fecal suspension was inoculated into the fermenting tank. Under the anaerobic condition, the number of four dominant bacteria in the medium was recorded by the plate dilution counting method. The growth curves of the four bacteria were plotted and the logarithmic growth period was calculated. On the basis, the optimized continuous culture process, including dilution rate (0.05h~ (-1), 0.07h~ (-1) and 0.09h~ (-1)) and the time of continuous culture (5h, 7h and 9h), was optimized by using the number of four dominant bacteria and three main short chain fatty acids in the culture medium, and the simulation system of human intestinal microecological chemostat was established. A 30 d experiment was conducted to evaluate the stability and reliability of the simulation system.
Batch culture showed that the logarithmic growth period of the four dominant bacteria was not the same, but it all contained the 3-8h. in the 0.07h~ (-1) condition, the system reached the steady state before the continuous culture, so 0.07h~ (-1) was a relatively suitable dilution rate. When the 7h was transferred to continuous culture, the system reached the steady state earlier than the other two conditions and the steady state system. The number of bacterial flora and SCFA concentration in the system is also the highest, so 7h is a relatively suitable time to transfer to continuous culture time.30d test shows that in the system of 9-30d, the difference of the number of four representative bacteria and the concentration of three kinds of SCFA is not significant, and the stability of the system culture conditions (such as temperature, stirring speed, pH value, etc.) is also better. After reaching steady state, the number of four dominant bacteria and three kinds of SCFA concentration in the culture system decreased slightly compared with the fecal phase before inoculation, but all were within the normal physiological parameters of the reported healthy people.
The experimental results show that the simulation system of human intestinal microecological chemostat has been successfully established by exploring and optimizing the dilution rate and continuous culture time. The system has good stability and reliability and can simulate the intestinal microflora of healthy people. This study is the first time from the number of bacteria group and metabolite concentration. The stability and reliability of the chemostat simulation system are evaluated by two angles, which fill the blank of the domestic research. This study also solves the problems of uneven agitation, unstable liquid level and complicated operation, which lead to the relative instability of the simulation system.
The establishment of the chemostat simulation system can provide a platform for the safety evaluation of the influence of antimicrobial residues on the intestinal flora, and also provide more scientific methods and ways for the safety evaluation and drug metabolism research of new drugs, functional foods and so on. It can ensure the safety of human food and medicine and promote the development of related disciplines. Important meaning.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R-331

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