比色法快速检测食品中大肠菌群的研究

发布时间：2018-05-23 14:09

本文选题：大肠菌群 + 比色法　；参考：《吉林大学》2012年硕士论文

【摘要】：随着致病菌引起的食源性食物中毒事件频繁发生，国内对食品中微生物快速检测方法的研究也越来越重视，力求改善现行传统检测方法的不足，以满足快速、准确检测食品质量的需要。大肠菌群是食品检验中最常用的指标之一。大肠菌群是指在37℃环境下能发酵乳糖，产酸产气的一群需氧或兼性厌氧的革兰氏阴性无芽胞杆菌。目前普遍采用的大肠菌群的检测方法为多管发酵法。虽然传统的多管发酵法检测大肠菌群数具有准确、可靠等优点，但是在食品和水质的监测过程中，或在检测样品多和检测时间紧的情况下，由于其检测时间较长，已越来越不能满足实际监测的需要了。本文主要利用大肠菌群的培养共性，对基于颜色特征识别的大肠菌群的快速计数法进行研究。本文主要研究内容和成果如下： 1．对比色法检测大肠菌群的显色培养基配方进行设计，确定了以月桂基磺酸盐胰蛋白胨培养基(LST)为基础，通过添加营养物质、酸碱指示剂、抑菌剂、表面活性剂等组分进行优化，筛选出显色培养基的最佳配方为：LST4.56g、溴甲酚紫0.03g、1.5%乳糖溶液0.1mL(培养40min后加入)、吐温0.2%，100mL无菌水。同时设计半固体琼脂培养基，通过对比不同琼脂含量对汽包的截留情况，确定了琼脂含量0.8%时观察菌体产气效果最佳，确保判断大肠菌群阳性结果的准确性。 2．对大肠菌群数目与培养液颜色之间的关系进行研究，通过测量经大肠菌群发酵产酸后培养基的颜色信息，采用线性或非线性拟合方法建立大肠菌群数目与培养基颜色信息之间的模型，最终确定BP神经网络模型能够得到理想的预测结果，并建立了大肠菌群数目与颜色对应标准比色板，适用于食品现场安全监察检测。 3.对固体类食品、生活饮用水大肠菌群快速检测方法进行研究，最终提供了的比色法定量检测技术与检测结果如下： (1)检测技术：在无菌条件下准确称取25g固体样品，放入到装有50mL无菌生理盐水的锥形瓶中，置于摇床上160r/min振荡2min，混匀样品，用中速定量滤纸过滤样品匀液，再用50mL无菌生理盐水冲洗滤纸上残留的物质，收集滤液。无菌操作吸取6mL菌液通过滤膜进行浓缩，使菌体在过滤器中分布均匀，然后将微量移液管贴近滤膜吸取20μL菌液。准确称取4.56g月桂基磺酸盐胰蛋白胨培养基于100mL蒸馏水中，，加热溶解，加入0.03g溴甲酚紫指示剂和2%的吐温试剂，振荡摇匀后以棉塞封口，放入灭菌锅中121℃温度下灭菌20min。取出置于无菌工作台上，待溶液冷却至37℃时调节pH值6.99。用无菌微量移液器吸取1mL培养基放入1.5mL离心管中，加入浓缩后的待检液20μL，37℃恒温箱中培养120min。培养40min后取出置于工作台上，加入1.5%的乳糖溶液100μL，盖紧离心管的盖子，置于恒温箱中继续培养。培养120min后，取出离心管，观察发酵液的颜色，对照大肠菌群数-颜色标准比色版读取大肠菌群数目的估计值。 (2)检测结果：该法在120min内即可得到检测结果，且比色法快速检测大肠菌群结果与国标法检测结果的比值为1.21，二者无明显差别，但国标法检测需72h，该法能明显缩短检测时间，具有一定的灵敏性和真实性。本课题基于大肠菌群生物培养共性，改良了大肠菌群的培养基，建立大肠菌群数目与颜色对应模型，大大地缩短了检测时间，达到快速，准确，实时地报告检测结果。这对控制食品安全性起到重要作用，将对食品生产的时效性起到推动作用，同时也将带来一定的经济效益。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5

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