本文選題：大腸菌群 + 比色法��； 參考：《吉林大學》2012年碩士論文

【摘要】：隨著致病菌引起的食源性食物中毒事件頻繁發(fā)生，國內(nèi)對食品中微生物快速檢測方法的研究也越來越重視，力求改善現(xiàn)行傳統(tǒng)檢測方法的不足，以滿足快速、準確檢測食品質量的需要。大腸菌群是食品檢驗中最常用的指標之一。大腸菌群是指在37℃環(huán)境下能發(fā)酵乳糖，產(chǎn)酸產(chǎn)氣的一群需氧或兼性厭氧的革蘭氏陰性無芽胞桿菌。目前普遍采用的大腸菌群的檢測方法為多管發(fā)酵法。雖然傳統(tǒng)的多管發(fā)酵法檢測大腸菌群數(shù)具有準確、可靠等優(yōu)點，但是在食品和水質的監(jiān)測過程中，或在檢測樣品多和檢測時間緊的情況下，由于其檢測時間較長，已越來越不能滿足實際監(jiān)測的需要了。本文主要利用大腸菌群的培養(yǎng)共性，對基于顏色特征識別的大腸菌群的快速計數(shù)法進行研究。 本文主要研究內(nèi)容和成果如下： 1．對比色法檢測大腸菌群的顯色培養(yǎng)基配方進行設計，確定了以月桂基磺酸鹽胰蛋白胨培養(yǎng)基(LST)為基礎，通過添加營養(yǎng)物質、酸堿指示劑、抑菌劑、表面活性劑等組分進行優(yōu)化，篩選出顯色培養(yǎng)基的最佳配方為：LST4.56g、溴甲酚紫0.03g、1.5%乳糖溶液0.1mL(培養(yǎng)40min后加入)、吐溫0.2%，100mL無菌水。同時設計半固體瓊脂培養(yǎng)基，通過對比不同瓊脂含量對汽包的截留情況，確定了瓊脂含量0.8%時觀察菌體產(chǎn)氣效果最佳，確保判斷大腸菌群陽性結果的準確性。 2．對大腸菌群數(shù)目與培養(yǎng)液顏色之間的關系進行研究，通過測量經(jīng)大腸菌群發(fā)酵產(chǎn)酸后培養(yǎng)基的顏色信息，采用線性或非線性擬合方法建立大腸菌群數(shù)目與培養(yǎng)基顏色信息之間的模型，最終確定BP神經(jīng)網(wǎng)絡模型能夠得到理想的預測結果，并建立了大腸菌群數(shù)目與顏色對應標準比色板，適用于食品現(xiàn)場安全監(jiān)察檢測。 3.對固體類食品、生活飲用水大腸菌群快速檢測方法進行研究，最終提供了的比色法定量檢測技術與檢測結果如下： (1)檢測技術：在無菌條件下準確稱取25g固體樣品，放入到裝有50mL無菌生理鹽水的錐形瓶中，置于搖床上160r/min振蕩2min，混勻樣品，用中速定量濾紙過濾樣品勻液，再用50mL無菌生理鹽水沖洗濾紙上殘留的物質，收集濾液。無菌操作吸取6mL菌液通過濾膜進行濃縮，使菌體在過濾器中分布均勻，然后將微量移液管貼近濾膜吸取20μL菌液。準確稱取4.56g月桂基磺酸鹽胰蛋白胨培養(yǎng)基于100mL蒸餾水中，，加熱溶解，加入0.03g溴甲酚紫指示劑和2%的吐溫試劑，振蕩搖勻后以棉塞封口，放入滅菌鍋中121℃溫度下滅菌20min。取出置于無菌工作臺上，待溶液冷卻至37℃時調(diào)節(jié)pH值6.99。用無菌微量移液器吸取1mL培養(yǎng)基放入1.5mL離心管中，加入濃縮后的待檢液20μL，37℃恒溫箱中培養(yǎng)120min。培養(yǎng)40min后取出置于工作臺上，加入1.5%的乳糖溶液100μL，蓋緊離心管的蓋子，置于恒溫箱中繼續(xù)培養(yǎng)。培養(yǎng)120min后，取出離心管，觀察發(fā)酵液的顏色，對照大腸菌群數(shù)-顏色標準比色版讀取大腸菌群數(shù)目的估計值。 (2)檢測結果：該法在120min內(nèi)即可得到檢測結果，且比色法快速檢測大腸菌群結果與國標法檢測結果的比值為1.21，二者無明顯差別，但國標法檢測需72h，該法能明顯縮短檢測時間，具有一定的靈敏性和真實性。 本課題基于大腸菌群生物培養(yǎng)共性，改良了大腸菌群的培養(yǎng)基，建立大腸菌群數(shù)目與顏色對應模型，大大地縮短了檢測時間，達到快速，準確，實時地報告檢測結果。這對控制食品安全性起到重要作用，將對食品生產(chǎn)的時效性起到推動作用，同時也將帶來一定的經(jīng)濟效益。
[Abstract]:With the frequent occurrence of food borne food poisoning caused by pathogenic bacteria, more and more attention has been paid to the research of rapid microbial detection methods in food, in order to improve the shortcomings of the existing traditional detection methods so as to meet the needs of rapid and accurate detection of food quality. Coliform is one of the most commonly used indexes in food inspection. Group refers to a group of aerobic or facultative anaerobic bacillus free bacillus that can ferment lactose and produce gas at 37 centigrade. The commonly used methods for detecting coliform are multi tube fermentation. Although the traditional multi tube fermentation method has the advantages of accurate and reliable detection of the number of coliform bacteria, it is monitored in food and water quality. In the process, in the case of many samples and tight detection time, because of its long detection time, it has become more and more unable to meet the needs of actual monitoring. This paper mainly uses the common culture of coliform group to study the fast counting method of coliform colony based on color feature recognition.
The main contents and results of this paper are as follows:
1. the color culture medium of coliform group was designed by contrast color method. The optimum formula was selected by adding nutrients, acid base indicator, bacteriostat and surface active agent on the basis of LST. The best formula was LST4.56g, bromocresol violet 0.03g, 1.. 5% lactose solution 0.1mL (culture 40min after adding), Twain 0.2%, 100mL aseptic water. Meanwhile, the semi solid agar medium was designed. By comparing the retention of different agar content to the drum, it was determined that when the agar content was 0.8%, the best effect of the bacteria was observed and the accuracy of the positive results of the coliform group was judged.
2. the relationship between the number of coliform group and the color of the culture medium was studied. By measuring the color information of the culture medium after the coliform fermented, the linear or nonlinear fitting method was used to establish the model between the number of coliform group and the color information of the medium. Finally, the BP neural network model could be predicted to be ideal. Results the collocation of coliform number and color corresponding standard colorimetric plate was established, which is suitable for food field safety monitoring.
3. the rapid detection method for coliform bacteria in solid food and drinking water was studied. Finally, the colorimetric quantitative detection technology and test results were provided as follows:
(1) detection technology: accurately named 25g solid sample under aseptic condition, put into conical bottle with 50mL aseptic saline, 160r/min oscillating 2min on the rocking bed, mix the sample, filter the sample with medium speed quantitative filter paper, and then use 50mL aseptic saline to rinse the residue on the filter paper and collect the filtrate. The aseptic operation absorbs 6mL. The bacteria solution was concentrated in the filter membrane, and the bacteria were evenly distributed in the filter. Then the micropipette was close to the filter membrane to absorb 20 L bacteria liquid. 4.56g lauryl sulfonate tryptone was accurately called to be cultured in the distilled water of 100mL, heated and dissolved, added to the 0.03g bromino cresol violet indicator and 2% Twain reagent. In the sterilization pot, the sterilized 20min. was removed at 121 C and placed on the aseptic worktable. When the solution was cooled to 37 centigrade, the pH value was adjusted and the 1mL medium was sucked into the 1.5mL centrifuge tube by the aseptic micropipette, and the concentrated liquid was added to the 1.5mL centrifuge tube, and the concentration of the treated liquid was 20 u L, and the incubator of 120min. was cultured on the worktable, and 1.5% milk was added after the incubating of the 120min. in the incubator at 37. The sugar solution was 100 u L, and the lid of the centrifuge tube was tightened and kept in the incubator. After 120min, the centrifuge tube was taken out to observe the color of the fermentation broth, and the number of coliform standard colorimetric version was compared to the number of coliform standard colorimetry.
(2) the result of detection: the method can get the result in 120min, and the ratio of colorimetric method to detect the result of coliform group and national standard method is 1.21, and there is no obvious difference between the two, but the national standard method is 72h. This method can obviously shorten the detection time, and it has a certain sensitivity and authenticity.
This subject is based on the commonness of coliform group biological culture, improves the medium of coliform group, establishes the number of coliform group and the model of color correspondence, greatly shortens the detection time, and achieves the rapid, accurate and real-time report of the detection results. This plays a important role in controlling the safety of food and will play an important role in the timeliness of food production. At the same time, it will also bring some economic benefits.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R155.5

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