Design of Milk Microbiological Detector

In the past two years, some foreign technology companies have successively developed relevant testing instruments for rapid detection of microorganisms in food and dairy products, and have established agents in my country. The most representative one is the Pi-102 food bacteria rapid tester produced by Hygiena in the United States. This food bacteria content rapid tester mainly uses the ATP bioluminescence method, combined with high-sensitivity photomultiplier tubes and special reagents, to quickly estimate the total number of microorganisms contained in food samples (after pretreatment) within 10 minutes. The test result data is matched with the corresponding national standards, and the microbial situation in the food sample can be preliminarily judged, such as whether it exceeds the standard. ATP is the abbreviation of adenosine triphosphate, which is an energy supply molecule in cells. The Pi-102 food bacteria rapid tester obtains the light unit reading by detecting the luminescence reaction of ATP and luciferase in the sample. After conversion, the total level of ATP can be estimated, and then the total number of microorganisms in the sample can be further indirectly judged to provide a reference for on-site inspection of food hygiene supervision. The sales price of this tester in China is RMB 79,000. However, because the ATP bioluminescence reaction is affected by various factors (temperature, acidity, alkali, dilution concentration, etc.), the results deviate from the CFU (colony forming unit) obtained by conventional culture. Therefore, the test recommends that users set a critical value for reference.

Secondly, BactoCount IBC-M is the latest semi-automatic milk bacteria counter launched by BENTLEY in the United States. The instrument is designed for small and medium-sized laboratories. The rapid detection results make it a tool for milk processing plants or milk product laboratories, thus filling the gap in the rapid detection of microorganisms in milk sources. The detection steps and principles of this instrument are first culture (sample preparation), that is, adding a culture reagent composed of a purification buffer, a hydrolyzing protease and a fluorescent marker liquid to the milk sample to dissolve the somatic cells in the milk sample, stabilize fat globules and proteins, penetrate bacteria and contaminate DNA. During the culture process, the fluorescent marker liquid can quickly penetrate into all bacteria with double-layer nucleic acids. At the same time, the ultrasonic degrader can chemically degrade interfering particles, disintegrate the remaining bacterial colonies and reduce background fluorescence. The sales price of this detector in China is RMB 720,000.

Another is the BactoScan FC series milk bacteria count rapid analyzer from Foss, Denmark. With this detection system, users can obtain the total bacteria count in raw milk within one minute.

The total number of bacteria can provide fast and reliable data for farms and dairy processing companies. With this fast and accurate information, farms can improve their hygiene conditions in a timely manner and improve the quality of the products they sell. In particular, dairy processing plants can use its fast advantage to effectively test the raw materials entering the plant in real time, so as to keep raw milk with unqualified bacterial indicators out of the processing plant, thereby avoiding losses to processing companies. Reliable data can provide a fair and comprehensive analysis method for farms and dairy plants to price according to quality.

At present, Flow Cytometry (flow cell counting principle/counting and identifying the characteristics of cells, bacteria or particles in flow) technology has been widely used in biochemistry and medical science. Its principle is to dye the cells/particles to be measured and place them in a suspended liquid, and then force the cells/particles to pass through a very narrow gap/pipe one by one. At the same time, a special light beam is emitted to them, so that each stained cell/particle also emits a response light beam pulse signal when passing through the measurement point, and then the pulse signal is recorded using electronic technology. The sales price of this detector in China is 1.4 million yuan.

2 Information collection and amplification circuit design

In recent years, experts in the fields of microbiology, biomedicine, physics, and electronic information have conducted extensive and in-depth research on the detection of pesticide residues, harmful chemical components, and harmful microorganisms in food, and published a large number of relevant academic papers. The representative bioelectrochemical method for microbial detection is the article "Study on Biosensors for Microbial Living Cell Detection" published by Cai Haobin in Huaxia Medicine in 2000 and the article "Study on a Bioelectrochemical Method for Microbial Detection" published by Lu Zhiyuan et al. in the Journal of Sensor Technology in 2005. However, there are no reports on the actual application of microbial detection in this area in China.

Based on the previous research, this paper sampled and processed the experimental data in the relevant literature, and designed and produced a rapid microbial detector. Table 1 lists the experimental data of fresh milk bacteria content and sensor at 25℃ given by Lu Zhiyuan et al. in their literature.

As can be seen from Table 1, the current and voltage output by the sensor are very weak, so errors may occur in the analog-to-digital conversion and signal processing of the information, so an amplifier circuit with micro-current amplification as shown in Figure 1 should be designed. The amplifier circuit uses ICL7650 with high gain, high common mode rejection ratio, small offset and low drift as an operational amplifier, and considers factors such as phase compensation, power supply fluctuations and interference filtering. The amplifier has high input impedance and low output impedance, and the closed-loop amplification factor is 50. The signal output by the biological battery box can be effectively amplified by the micro-current amplifier.

The detector can start collecting current data by clicking the Start button in the figure, and then directly obtain the corresponding number of bacteria in the milk through the function calculation in the program. When the Stop button is clicked, the curve stops, and the corresponding current value and bacterial number numerical display are the instantaneous values ​​when the button is clicked. Figure 3 shows the actual picture of the detector.

3 Conclusion

The microbial detector composed of the biological battery box and the software system introduced in this article can use Matlab to fit and process the curves obtained from repeated measurements, so as to obtain the final curve of the sensor output voltage and the bacterial content. According to the curve data, a program can be written to obtain the corresponding data of the sensor output voltage and the bacterial content of dairy products. Then the tester is used to measure the bacterial content of aseptic bagged fresh milk to verify the effectiveness of the electrochemical biosensor. The data detection error of the detector is 5%, so its related technologies still need to be improved and improved. However, the detector has greatly improved the speed and efficiency of microbial detection. Moreover, the data output by the test is intuitive and clear, which changes the traditional method of manual counting of bacteria or statistical counting by microscopic observation.