Device and method for rapidly detecting escherichia coli in milk powder

Abstract

The invention provides a device for rapidly detecting escherichia coli in milk powder, which comprises a sample plate, a separation membrane, a sealing ring, a receiving tube and a sealing cover which are sequentially assembled from bottom to top, and also provides a method for rapidly detecting escherichia coli in milk powder, which comprises the following steps: preparing a milk powder sample by using sterile water and culturing to obtain a culture solution; separating acetic acid of escherichia coli in the culture solution, absorbing by using an absorption solution, observing color change of the absorption solution, and carrying out qualitative analysis; or shooting to obtain an absorption liquid image, analyzing the RGB three-primary color composition of the absorption liquid image through image recognition software, and obtaining a quantitative analysis result according to a linear relation curve between the G / B value or (R + G) / B value and the escherichia coli concentration. The rapid detection device for the escherichia coli in the milk powder is portable, small in size and low in cost, and can meet the field detection requirement; escherichia coli metabolizes carbohydrates to generate acetic acid, and the color of the absorption liquid is changed by separating the acetic acid, so that the escherichia coli in the milk powder is rapidly detected.

Description

Technical Field

[0001] The invention relates to the field of food microorganism detection, and in particular to a device and method for rapid detection of Escherichia coli in milk powder. Background Art

[0002] Escherichia coli, also known as Escherichia coli, was discovered by Escherich in 1885. Escherichia coli is a Gram-negative short rod with blunt ends, no spores and motile. Its main parasitic site is the large intestine of organisms, accounting for about 1% of intestinal bacteria. Escherichia coli is a conditional pathogen. Under certain conditions, Escherichia coli causes gastrointestinal infections in humans or animals, mainly caused by specific fimbriae antigens, pathogenic toxins, etc. In addition to gastrointestinal infections, it can also cause urinary tract infections, arthritis, meningitis, and septicemic infections. Infection with Escherichia coli can lead to a wide range of clinical manifestations, including asymptomatic infection, mild diarrhea, or serious diseases (such as hemorrhagic colitis and hemolytic uremic syndrome).

[0003] According to WHO statistics, more than 600 million food safety incidents are caused by E. coli each year, which also shows that E. coli contamination is a prominent problem in food safety. Therefore, in order to ensure food safety, the detection of E. coli has become a necessary test item for food microbiological testing.

[0004] Infant formula milk powder is made from milk or milk protein products as the main raw materials. According to the nutritional needs of infants and young children, the ingredients are as similar to breast milk as possible. After nutritional proportioning, nutrients beneficial to the growth of infants and young children are added to produce and process the powdered products, which can meet the normal nutritional and energy needs of infants and young children. Infant milk powder is rich in nutrients, so it is considered to be a good culture medium that can support bacterial growth and has the potential risk of exposure to bacteria. Infants and young children are a special group. Due to their imperfect digestive system and immature metabolic system, they are easily infected by microorganisms. Therefore, infant formula milk powder has a great impact on the health of infants and young children, so it is particularly important to control its microbial contamination to ensure the quality and safety of milk powder.

[0005] At present, the detection of Escherichia coli in milk powder mainly relies on GB 19644-2010. Although it has high accuracy and reliable results, the steps are cumbersome, the experimental cycle is long, it is difficult to achieve the purpose of rapid detection, and the detection limit is 10 cfu / g, with relatively high requirements. In recent years, many rapid detection technologies for Escherichia coli have emerged, such as immunomagnetic bead method, immunochromatographic gold test strip method, gene chip technology, flow cytometry analysis technology, loop-mediated isothermal amplification technology, biosensor technology, etc. However, these technologies also have certain deficiencies, such as high cost, complex sample pretreatment, high requirements for inspection technicians, high requirements for detection and analysis equipment, etc. Therefore, the simplification of sample pretreatment and detection means has become a bottleneck problem for the rapid analysis of Escherichia coli. Summary of the Invention

[0006] Based on this, in order to achieve the rapid detection of Escherichia coli in milk powder, the present invention provides a rapid detection device for Escherichia coli in milk powder. By utilizing the characteristic that Escherichia coli metabolizes carbohydrates to generate acetic acid, a thermal-assisted gas membrane separation device is constructed to separate acetic acid, and a rapid qualitative and quantitative detection and analysis method for Escherichia coli in milk powder is provided.

[0007] In the first aspect of the present invention, a rapid detection device for Escherichia coli in milk powder is provided, including a sample plate, a separation membrane, a sealing ring, a receiving tube and a sealing cover; a plurality of stepped grooves are provided on the sample plate, and each stepped groove forms a sample pool and a connecting part arranged from bottom to top. The separation membrane covers the sample pool, the sealing ring is arranged on the separation membrane, one end of the receiving tube is detachably and hermetically connected to the connecting part, and the end presses the sealing ring against the sample pool, and the other end of the receiving tube is detachably and hermetically connected to the sealing cover.

[0008] The present invention is used for detecting Escherichia coli in milk powder. By utilizing the characteristic that Escherichia coli metabolizes carbohydrates to produce acetic acid, a device capable of separating acetic acid is designed. The rapid detection device for Escherichia coli in milk powder described in the present invention includes a sample plate, a separation membrane, a sealing ring, a receiving tube, and a sealing cover. The sample plate includes a sample pool and a connecting part arranged from bottom to top. The separation membrane, the sealing ring, the receiving tube, and the sealing cover are sequentially connected on the sample pool in sequence. When in use, the milk powder is configured into a test solution and placed in the sample pool. If Escherichia coli is contained in the milk powder, the Escherichia coli will metabolize the milk powder to produce acetic acid. Since acetic acid is volatile and easily penetrates through the separation membrane into the receiving tube, the separation of acetic acid is achieved. By absorbing acetic acid with a corresponding absorbent solution to produce observable or detectable changes, Escherichia coli can be detected. Heating can accelerate the separation of acetic acid. The boiling point of acetic acid (117.9 °C) is relatively high, and in this device, a sealing ring is added to the separation membrane to further enhance the airtightness and high temperature and high pressure resistance performance of the device. The end of the receiving tube presses the sealing ring against the sample pool. During the heating process, even if the water and acetic acid in the sample tube boil and the air pressure increases sharply, the solution will not be ejected and the separation membrane will not come off. Therefore, it is suitable for the rapid separation of acetic acid, and further realizes the rapid detection of Escherichia coli. Due to the good airtightness of this device, it can also be used in the in-situ detection mode. Since the receiving liquid is isolated from the external environment, it can avoid absorbing carbon dioxide in the air and producing false positive results. In addition, the design of the thermal-assisted array gas membrane separation device described in the present invention realizes miniaturization, is convenient to carry, and can meet the requirements of on-site detection.

[0009] As a preferred solution, the separation membrane is a waterproof and breathable membrane. The waterproof and breathable membrane is impermeable to water but allows acetic acid to penetrate, achieving the purpose of separating acetic acid and water.

[0010] As a preferred solution, the separation membrane is a polytetrafluoroethylene (PTFE) membrane, making it have more excellent high temperature and high pressure resistance performance. Preferably, all components in the detection device are made of polytetrafluoroethylene material.

[0011] As a preferred solution, the connecting part is threadedly connected to the receiving tube, and the receiving tube is threadedly connected to the sealing cover; the connecting part is provided with a first threaded interface, the two ends of the receiving tube are respectively provided with a second threaded interface and a third threaded interface, and the sealing cover is provided with a fourth threaded interface; the second threaded interface matches the first threaded interface, and the fourth threaded interface matches the third threaded interface. In order to enhance the airtightness of the device, fine threads are provided on the sample pool, the receiving tube, and the sealing cover, making them tightly connected like screws and nuts.

[0012] As a preferred solution, a plurality of sample pools are arranged in an array in the sample plate, which is convenient for simultaneous detection of a large batch.

[0013] In a second aspect of the present invention, there is also provided a method for rapid detection of Escherichia coli in milk powder, comprising the following steps:

[0014] Prepare a milk powder sample to be tested with sterile water and culture it at 37 °C to obtain a culture solution of the milk powder to be tested;

[0015] Add the culture solution of the milk powder to be tested into the sample pool of the above detection device, sequentially place and install the separation membrane, the sealing ring and the receiving tube, drop an absorption liquid onto the separation membrane, the absorption liquid is an acid-base indicator that can produce a color change after absorbing an acidic gas and the color change range is at pH = 5-9, and then install the sealing cover;

[0016] Observe the color change of the absorption liquid for qualitative analysis.

[0017] This method utilizes the fact that Escherichia coli metabolizes carbohydrates to produce acetic acid. Due to the volatility of acetic acid, acetic acid is separated through the separation membrane and enters the absorption liquid to cause a color change in the absorption liquid, so as to quickly judge whether Escherichia coli exists in the sample, and it is suitable for on-site rapid detection.

[0018] A method for rapid detection of Escherichia coli in milk powder, comprising the following steps:

[0019] Prepare milk powder solutions containing Escherichia coli with different concentrations using sterile water as standard samples of Escherichia coli bacterial solutions, and culture them at 37 °C to obtain multiple standard sample culture solutions;

[0020] Add each of the standard sample culture solutions into the sample pool of the detection device in the first part, and only add one standard sample to be tested to each sample pool. Sequentially place and install the separation membrane, the sealing ring and the receiving tube, drop an absorption liquid onto the separation membrane, the absorption liquid is an acid-base indicator that can produce a color change after absorbing an acidic gas and the color change range is at pH = 5-9, and then install the sealing cover;

[0021] Heat the sample plate, and immediately take a picture of the absorption liquid after the heating ends to obtain an image of the absorption liquid;

[0022] Analyze the RGB three-primary color composition of the absorption liquid image through image recognition software to obtain the R value, G value, and B value, and construct a standard curve of G / B value - Escherichia coli concentration or (R + G) / B value - Escherichia coli concentration;

[0023] Prepare a milk powder sample to be tested with sterile water and culture it at 37 °C to obtain a culture solution of the milk powder to be tested;

[0024] Add the culture solution of the milk powder to be tested into the sample pool of the detection device in the first part, sequentially place and install the separation membrane, the sealing ring and the receiving tube, drop an absorption liquid onto the separation membrane, and then install the sealing cover;

[0025] Heat the sample plate, and immediately take a picture of the absorbent solution after the heating is completed to obtain an image of the absorbent solution;

[0026] Analyze the RGB three - primary - color composition of the absorbent - solution image through image - recognition software to obtain the R value, G value, and B value. Substitute the G / B value into the G / B value - Escherichia coli concentration standard curve or substitute the (R + G) / B value into the (R + G) / B value - Escherichia coli concentration standard curve to obtain the Escherichia coli concentration in the milk - powder sample to be tested.

[0027] This method utilizes the fact that Escherichia coli metabolizes carbohydrates to produce acetic acid. By separating acetic acid, the color of the absorbent solution changes. Then, the absorbent - solution image is analyzed and processed. According to the color composition of the absorbent solution, the acetic - acid content can be judged. And the amount of acetic - acid production has a linear relationship with the Escherichia coli concentration, so as to quantitatively analyze the Escherichia coli concentration in the sample. Through quantitative analysis of multiple groups of standard samples, the G / B value - Escherichia coli concentration and (R + G) / B value - Escherichia coli concentration standard curves are obtained. The standard curves have high accuracy and can reduce errors in detection and analysis. In practical applications of this method, a smartphone can be used to complete the photographing and analysis of the absorbent solution, realizing the rapid quantitative analysis of Escherichia coli in milk powder. Compared with the prior art, it is more suitable for on - site rapid detection.

[0028] As a preferred solution, the heating temperature of the sample plate is 110 °C and the heating time is 15 min, or the heating temperature is 37 °C and the heating time is 24 h. Heating at 110 °C for 15 min can obtain more accurate quantitative - detection results. If the heating temperature is too low and the time is too short, the acetic acid in the culture solution volatilizes incompletely, affecting the quantitative - detection results. If the heating temperature is too high and the time is too long, not only is the energy consumption large, but after the acetic acid in the culture solution enters the absorbent solution, it is easy to volatilize again, resulting in the absorbent solution being unable to completely absorb the acetic acid produced in the culture solution, affecting the quantitative - detection results. If only qualitative analysis is required, it can be heated at 37 °C for 24 h. In - situ analysis at this temperature and time is more suitable for qualitative analysis.

[0029] As a preferred solution, the incubation time of the milk powder sample to be tested and the Escherichia coli standard sample before adding them into the sample cell is 15 - 24 h. When the incubation time is within 15 - 24 h, the Escherichia coli is in the logarithmic growth phase. In this phase, the Escherichia coli shows a stable geometric progression growth. There are significant differences in the amount of acetic acid produced by Escherichia coli at different concentrations, making the color change of the absorbent solution the most obvious, thereby improving the accuracy of the quantitative analysis results. If the incubation time is too short, the Escherichia coli is in the lag growth phase, and less acetic acid is produced during this phase, resulting in an unclear color change of the absorbent solution and inaccurate quantitative analysis results. If the incubation time is too long, the Escherichia coli is in the stationary phase or the decline phase, and the concentration of Escherichia coli is roughly the same during this phase, and the continuously accumulating acetic acid causes the colors of the absorbent solutions to tend to be similar, making it impossible to perform quantitative analysis at this time. Therefore, it is necessary to strictly control the incubation time to make the Escherichia coli in the logarithmic growth phase.

[0030] As a preferred solution, the sterile water is prepared as follows: Prepare a solution according to the mass ratio of cholate, phosphate buffer with a pH of 6.86, and deionized water of 1:2:200, and then seal and sterilize it to obtain sterile water. Since cholate has an inhibitory effect on most bacteria, while Escherichia coli is not inhibited by cholate, using cholate to prepare sterile water can avoid the interference of most other bacteria and ensure the accuracy of the detection results.

[0031] This application utilizes the characteristic that Escherichia coli metabolizes carbohydrates to produce acetic acid, constructs a gas membrane separation device to separate it and then conducts detection and analysis, reducing the sample treatment process, shortening the treatment time, achieving rapid treatment, and having a low detection limit. Description of the Drawings

[0032] Figure 1 is a schematic structural diagram of the sample plate of the thermal-assisted array gas membrane separation device, including a top view, a front view, and a left view;

[0033] Figure 2 is a schematic structural diagram of the receiving tube of the thermal-assisted array gas membrane separation device, including a top view, a side view, and a three-dimensional view;

[0034] Figure 3 is a schematic structural diagram of the sealing cover of the thermal-assisted array gas membrane separation device, including a bottom view, a side view, and a three-dimensional view;

[0035] Figure 4 is a schematic diagram of the combination of each component of the thermal-assisted array gas membrane separation device;

[0036] Figure 5 is a linear relationship diagram between the G / B value of the smartphone sensing colorimetry method and the Escherichia coli concentration in Example 4;

[0037] Figure 6It is the linear relationship diagram of the (R+G) / B value of the smartphone sensing colorimetry method and the concentration of Escherichia coli in Example 4;

[0038] Figure 7 It is the linear relationship diagram of the gray value channel of the smartphone sensing colorimetry method in Example 4;

[0039] Figure 8 It is the HPLC diagram of the standard sample of Escherichia coli bacterial solution after 0 h of culture in Example 5;

[0040] Figure 9 It is the HPLC diagram of the standard sample of Escherichia coli bacterial solution after 24 h of culture in Example 5;

[0041] Figure 10 It is the linear relationship diagram of the absorption peak area of the HPLC method of the standard sample of Escherichia coli bacterial solution and the concentration of Escherichia coli in Example 5;

[0042] Wherein: 1-sample plate, 12-sample cell, 14-connection part, 2-separation membrane, 3-sealing ring, 4-receiving tube, 5-sealing cap, 61-first thread interface, 62-second thread interface, 63-third thread interface, 64-fourth thread interface. Detailed implementation manners

[0043] To further understand the present invention, the present invention will be described in detail below in conjunction with embodiments and drawings. However, it should be noted that the embodiments and drawings do not constitute a limitation on the scope of protection required by the present invention.

[0044] Embodiment 1

[0045] A rapid detection device for Escherichia coli in milk powder, as Figure 4 shown, includes a sample plate 1, a separation membrane 2, a sealing ring 3, a receiving tube 4 and a sealing cap 5. The sample plate 1 is provided with a plurality of stepped grooves, and each stepped groove forms a sample cell 12 and a connection part 14 arranged from bottom to top; the separation membrane 2 covers the sample cell 12, and the sealing ring 3 is arranged on the separation membrane 2; the device is designed with fine threads, the connection part 14 is threadedly connected to the receiving tube 2, the receiving tube 4 is threadedly connected to the sealing cap 5, and the thread designs of the connection part 14, the receiving tube 4 and the sealing cap 5 make them tightly connected like screws and nuts, greatly enhancing the high-pressure resistance performance of the device; the separation membrane 2 made of polytetrafluoroethylene (PTFE) material has excellent high-temperature and high-pressure resistance performance, the sealing ring 3 is added to the separation membrane 2, pressed by the receiving tube 4, and abuts against the sample cell 12, further increasing the airtightness and high-pressure resistance of the device.

[0046] As Figure 1As shown, the overall dimensions of the sample plate 1 are length A = 170 mm, width B = 130 mm, and height C = 20 mm. The sample plate 1 is provided with 48 evenly distributed stepped grooves, and the depth of each stepped groove is 15 mm, forming a sample cell 12 and a connecting part 14 arranged from bottom to top. The connecting part 14 is provided with a right-handed threaded interface 61 of M10×0.5. The right-handed threaded interface 61 is an internal thread, with an outer diameter of 10 mm, an inner diameter of 7 mm, and a depth of 3 mm.

[0047] As Figure 2 shown, the length L1 of the receiving tube 4 is 12 mm, the outer diameter is 10 mm, the inner diameter is 7 mm, and both ends are respectively designed with a left-handed threaded interface 63 and a right-handed threaded interface 62 of M10×0.5. The right-handed threaded interface 62 is an external thread, with an outer diameter of 10 mm, an inner diameter of 7 mm, and a depth H2 of 3 mm; the left-handed threaded interface 63 is an external thread, with an outer diameter of 10 mm, an inner diameter of 7 mm, and a depth H1 of 3 mm.

[0048] As Figure 3 shown, the length L2 of the sealing cap 5 is 14 mm, the outer diameter is 12 mm, and one end is designed with a left-handed threaded interface 64 of M10×0.5. The left-handed threaded interface 64 is an internal thread, with an outer diameter of 10 mm, an inner diameter of 7 mm, and a depth of 3 mm.

[0049] The combined diagram of the device is as Figure 4 shown. During assembly, from bottom to top are the sample plate 1, the separation membrane 2, the sealing ring 3, the receiving tube 4, and the sealing cap 5. The assembly process is as follows: cover the separation membrane 2 on the sample cell 12, then add the sealing ring 3, then press the receiving tube 4 against the sealing ring 3, and install and fix it on the sample plate 1 in a right-handed rotation to connect with the connecting part 14. Finally, install and fix the sealing cap 5 on the receiving tube 4 in a left-handed rotation to complete the assembly process. The core of the device is the separation membrane 2, with a diameter of 10 mm and a thickness of 0.3 mm.

[0050] Example 2

[0051] A colorimetric method for rapid detection of Escherichia coli in milk powder: Weigh 1.0 g of porcine bile salt and 2.0 g of phosphate buffer (pH = 6.86) into a 250 mL conical flask, dissolve with 200 mL of deionized water, seal with kraft paper, and place in a high-pressure steam sterilizer for sterilization at 121 °C for 20 min to obtain sterile water. Weigh 4 portions of 1.0 g of milk powder into 10 mL centrifuge tubes. Among them, 3 portions are added with Escherichia coli mother liquor, and then sterile water is added to 5.0 mL to respectively prepare bacterial solutions with concentrations of pOD600 = 3, pOD600 = 4, and pOD600 = 5, and the other 1 portion is used as a blank medium.

[0052] Put the bacterial solutions and blank medium of the above concentrations as Escherichia coli bacterial solution samples into an incubator at 37 °C for culturing for 24 hours.

[0053] After the cultivation, qualitative analysis was performed using the Escherichia coli rapid detection device described in Example 1. First, 300 μL of Escherichia coli broth samples (concentrations were blank medium, pOD600 = 3, pOD600 = 4, pOD600 = 5) were added to the sample pool, with three parallels for each concentration; then, a separation membrane and a sealing ring were placed on each sample pool, and the receiving tube was installed by rotating it to the right; next, 400 μL of bromocresol purple indicator was added to each sample receiving tube, and the sealing cap was installed by rotating it to the left. Finally, the sample plate was placed on the heating plate and run at 110 °C for 15 minutes.

[0054] The test results showed that the indicator color of the blank medium was purple, indicating that the sample did not contain Escherichia coli and did not metabolize glucose to produce acetic acid to change the indicator color. However, the indicator colors of the broth with concentrations of pOD600 = 3, pOD600 = 4, and pOD600 = 5 changed from purple to yellow, indicating the presence of Escherichia coli in the samples, and the metabolism of glucose to produce acetic acid changed the indicator color from purple to yellow. The results showed that the thermal-assisted array gas membrane separation device - colorimetry was successfully applied to the rapid qualitative analysis of Escherichia coli.

[0055] Example 3

[0056] An in-situ mode colorimetry for rapid detection of Escherichia coli in milk powder: Weigh 1.0 g of porcine bile salt and 2.0 g of phosphate buffer (pH = 6.86) into a 250 mL conical flask, dissolve with 200 mL of deionized water, seal with kraft paper, and place in a high-pressure steam sterilizer for sterilization at 121 °C for 20 min to obtain sterile water. Weigh 4 portions of 1.0 g of milk powder into 10 mL centrifuge tubes, add Escherichia coli mother liquor to 3 of them, and then add sterile water to 5.0 mL to prepare broths with concentrations of pOD600 = 3, pOD600 = 4, and pOD600 = 5 respectively, and the other 1 portion is used as the blank medium. Using the Escherichia coli broth samples of the above concentrations and the blank medium, qualitative analysis was performed using the Escherichia coli rapid detection device described in Example 1. First, 300 μL of Escherichia coli broth samples (concentrations were blank medium, pOD600 = 3, pOD600 = 4, pOD600 = 5) were added to the sample pool, with three parallels for each concentration; then, a separation membrane and a sealing ring were placed on each sample pool, and the receiving tube was installed by rotating it to the right; next, 400 μL of phenol red indicator was added to each sample receiving tube, and the sealing cap was installed by rotating it to the left. Finally, the sample plate was placed on the heating plate and in-situ qualitative analysis was performed at 37 °C.

[0057] Place the sample plate on the heating plate and run it at 37 °C for 24 h. The test results show that the indicator color of the blank medium is red, indicating that the sample does not contain Escherichia coli and does not metabolize the medium to produce acetic acid to cause a color change in the indicator. For the bacterial suspension with pOD600 = 3, the indicator color changes from red to yellow; for the bacterial suspension with pOD600 = 4, the indicator color changes from red to orange; for the bacterial suspension with pOD600 = 5, the indicator color changes from red to light red, indicating the presence of Escherichia coli in the sample. Furthermore, the bacteria metabolize the medium to produce acetic acid, causing a color change in the indicator, and achieving the effect of differentiating bacteria at different concentrations. The results show that the thermal-assisted array gas membrane separation device-in-situ mode colorimetry is successfully applied to the qualitative analysis of Escherichia coli.

[0058] Example 4

[0059] A smartphone sensing colorimetry method for rapid detection of Escherichia coli in milk powder: Weigh 1.0 g of porcine bile salt and 2.0 g of phosphate buffer (pH = 6.86) into a 250 mL conical flask, dissolve with 200 mL of deionized water, seal with kraft paper, and place it in an autoclave for sterilization at 121 °C for 20 min to obtain sterile water. Weigh 6 portions of 1.0 g of milk powder into 10 mL centrifuge tubes, add 0, 50, 100, 500, 1000 μL of 100 cfu / mL Escherichia coli standard solution in sequence, then add sterile water to 5.0 mL, and dissolve it thoroughly. Place the above Escherichia coli bacterial suspension and blank medium as the Escherichia coli bacterial suspension standard samples in an incubator at 37 °C for 15 hours.

[0060] After incubation, perform quantitative analysis using the Escherichia coli rapid detection device described in Example 1. First, add 300 μL of Escherichia coli bacterial suspension standard samples (concentrations are 0 cfu / g, 5 cfu / g, 10 cfu / g, 50 cfu / g, and 100 cfu / g respectively) to the sample pool, with three parallels for each concentration; then, place the separation membrane and sealing ring on each sample pool, and install the receiving tube by rotating it to the right; next, add 400 μL of bromocresol purple indicator to each sample receiving tube, and install the sealing cap by rotating it to the left. Finally, place the sample plate on the heating plate and run it at 110 °C for 15 minutes.

[0061] The detection results showed that the bromocresol purple indicator changed color from purple to light purple, gray, light yellow, and yellow as the concentration of Escherichia coli increased from low (0 cfu / g) to high (100 cfu / g). At the lowest concentration (5 cfu / g), the color of the indicator changed and the purple became lighter, indicating a detection limit of 5 cfu / g. The color of the indicator was sensed and identified by taking pictures using a smartphone sensing platform. The RGB three-primary colors of the indicator were analyzed by image recognition software. RGB represents the three colors of red, green, and blue respectively. Three channels, namely the G / B value, the (R + G) / B value, and the grayscale value, were constructed and numerical values were obtained. As Figure 5 shown, the G / B value showed a linear relationship with the Escherichia coli concentration in the range of 0 to 50 cfu / g (R 2 = 0.99121); as Figure 6 shown, the (R + G) / B value showed a linear relationship with the Escherichia coli concentration in the range of 0 to 50 cfu / g (R 2 = 0.98988); as Figure 7 shown, the grayscale value showed a relatively weak linear relationship with the Escherichia coli concentration in the range of 0 to 50 cfu / g (R 2 = 0.23088). The results indicated that based on the linear relationship between the G / B value and the Escherichia coli concentration, or the linear relationship between the (R + G) / B value and the Escherichia coli concentration as the standard curve, the thermal-assisted array gas film separation device - smartphone sensing colorimetry method could be applied to the rapid quantitative analysis of Escherichia coli, with a detection limit reaching 5 cfu / g.

[0062] Example 5

[0063] An HPLC method for the rapid detection of Escherichia coli in milk powder: Weigh 1.0 g of porcine bile salt and 2.0 g of phosphate buffer (pH = 6.86) into a 250 mL conical flask, dissolve with 200 mL of deionized water, seal with kraft paper, and place in a high-pressure steam sterilizer for sterilization at 121 °C for 20 min to obtain sterile water. Weigh 6 portions of 1.0 g of milk powder into 10 mL centrifuge tubes, sequentially add 0, 50, 100, 500, 1000 μL of 100 cfu / mL Escherichia coli standard solution, and then add sterile water to 5.0 mL and dissolve it thoroughly. The above Escherichia coli bacterial solution and blank medium were used as Escherichia coli bacterial solution standard samples and placed in an incubator at 37 °C for 24 hours.

[0064] After cultivation, quantitative analysis was performed using the Escherichia coli rapid detection device described in Example 1. First, 300 μL of standard Escherichia coli broth samples (concentrations of 0 cfu / g, 5 cfu / g, 10 cfu / g, 50 cfu / g, and 100 cfu / g respectively) were added to the sample pools, with three parallel groups for each concentration; then, a separation membrane and a sealing ring were placed on each sample pool, and the receiving tube was installed by rotating it to the right; next, 400 μL of deionized water was added to each sample receiving tube as the absorbent solution, and the sealing cap was installed by rotating it to the left. Finally, the sample plate was placed on the heating plate and run at 110 °C for 15 minutes.

[0065] The analysis results were detected by HPLC. As Figure 8 shown, after 0 hours of cultivation of Escherichia coli, no absorption peaks in the concentration range of 0 to 100 cfu / g were observed in the chromatogram; as Figures 9 - 10 shown, after 24 hours of cultivation of Escherichia coli, the absorbent solution had the same retention time as the standard acetic acid solution, and the absorbance peak area was linearly related to the Escherichia coli concentration in the range of 5 - 100 cfu / g (R 2 = 0.9880). The results successfully verified the mechanism that Escherichia coli produces acetic acid by metabolizing glucose, and indicated that the Escherichia coli rapid detection device described in the present invention was also applicable to the combination with the traditional HPLC method, achieving the purpose of rapid detection of Escherichia coli in milk powder, and the detection limit reached 5 cfu / g.

[0066] Example 6

[0067] A rapid detection method for Escherichia coli in milk powder: Weigh 1.0 g of porcine bile salt and 2.0 g of phosphate buffer (pH = 6.86) into a 250 mL conical flask, dissolve with 200 mL of deionized water, seal with kraft paper, and place in a high-pressure steam sterilizer for sterilization at 121 °C for 20 min to obtain sterile water. Weigh 1 portion of 0.5 g of Escherichia coli standard sample in milk powder (GBW(E)091095, 78 ± 13 cfu / g) into a 10 mL centrifuge tube, add 5.0 mL of sterile water, and then place in a constant temperature incubator at 37 °C for 15 hours.

[0068] After cultivation, quantitative analysis was performed using the Escherichia coli rapid detection device described in Example 1. First, 300 μL of the Escherichia coli standard sample was added to the sample pools, with two parallel groups; then, a separation membrane and a sealing ring were placed on each sample pool, and the receiving tube was installed by rotating it to the right; next, 400 μL of bromocresol purple was added to the first group of sample receiving tubes as the absorbent solution, while 400 μL of deionized water was added to the second group of sample receiving tubes as the absorbent solution, and the sealing cap was installed by rotating it to the left. Finally, the sample plate was placed on the heating plate and run at 110 °C for 15 minutes.

[0069] The detection results showed that the first group of bromocresol purple indicator changed color from purple to light yellow. After data processing, the G / B value was 1.7279. Substituting this value into the Figure 5 linear equation (G / B value - standard curve of Escherichia coli concentration) described in Example 4, its concentration was obtained as 39.5 cfu / g. The second group of deionized water absorbent was taken for HPLC analysis. After integration processing, the peak area of acetic acid was 12054. Substituting this value into the Figure 10 linear equation described in Example 5, its concentration was obtained as 39.3 cfu / g. Since Figure 5 and Figure 10 the sample weighing mass of the standard curve was 1.0 g and the weighing amount of the reference substance was 0.5 g, the results obtained after substituting into the standard curve needed to be multiplied by the dilution factor 2. Finally, the Escherichia coli concentration in the first group of standard samples was 39.5 cfu / g × 2 = 79.0 cfu / g, and the Escherichia coli concentration in the second group of standard samples was 39.3 cfu / g × 2 = 78.6 cfu / g. These results were consistent with the labeled amount of 78 ± 13 cfu / g of the standard sample, proving the accuracy of the method proposed in this application.

[0070] Comparative Example 1

[0071]

[0072] a. Including the sample pretreatment time

[0073] It can be seen from Comparative Example 1 that compared with the national standard, the detection limit of the present invention meets the requirements of GB 4789.3—2016 of the national standard (10 cfu / g), and the culture time of Escherichia coli is much lower than that of the national standard, and the detection time is equivalent to that of the national standard; compared with other existing technologies, the detection limit is lower, the culture time of Escherichia coli is similar, and the detection time is lower. The device and method for rapid detection of Escherichia coli in milk powder described in the present invention realizes the rapid detection of Escherichia coli in milk powder.

[0074] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A rapid detection device for Escherichia coli in milk powder, characterized in that, It includes a sample plate, a separation membrane, a sealing ring, a receiving tube and a sealing cover; several stepped grooves are provided on the sample plate, and each stepped groove forms a sample pool and a connecting part arranged from bottom to top. The separation membrane covers the sample pool, the sealing ring is arranged on the separation membrane, one end of the receiving tube is detachably and sealingly connected to the connecting part, and the end presses the sealing ring against the sample pool, and the other end of the receiving tube is detachably and sealingly connected to the sealing cover.

2. The rapid detection device for Escherichia coli in milk powder according to claim 1, wherein, The separation membrane is a waterproof and breathable membrane.

3. The rapid detection device for Escherichia coli in milk powder according to claim 2, characterized in that, The separation membrane is a polytetrafluoroethylene (PTFE) membrane.

4. The rapid detection device for Escherichia coli in milk powder according to claim 1, wherein The connecting part is threadedly connected to the receiving tube, and the receiving tube is threadedly connected to the sealing cover; the connecting part is provided with a first thread interface, the two ends of the receiving tube are respectively provided with a second thread interface and a third thread interface, and the sealing cover is provided with a fourth thread interface; the second thread interface matches the first thread interface, and the fourth thread interface matches the third thread interface.

5. The rapid detection device for Escherichia coli in milk powder according to claim 1, characterized in that, Several sample pools are arranged in an array in the sample plate.

6. A rapid detection method for Escherichia coli in milk powder, characterized in that It includes the following steps: Prepare a sample of the milk powder to be tested with sterile water and culture it at 37 °C to obtain a culture solution of the milk powder to be tested. Add the culture solution of the milk powder to be tested into the sample pool of the detection device according to any one of claims 1-5, place and install the separation membrane, the sealing ring and the receiving tube in sequence, drop an absorption liquid on the separation membrane. The absorption liquid is an acid-base indicator that can produce a color change after absorbing acidic gas and the color change range is at pH = 5-9, and then install the sealing cover. Observe the color change of the absorption liquid for qualitative analysis.

7. A rapid detection method for Escherichia coli in milk powder, characterized in that It includes the following steps: Prepare milk powder solutions containing Escherichia coli with different concentrations with sterile water as standard samples of Escherichia coli solutions, and culture them at 37 °C to obtain multiple standard sample culture solutions. Add each standard sample culture solution into the sample pool of the detection device according to any one of claims 1-5. Only one standard sample to be tested is added to each sample pool. Place and install the separation membrane, the sealing ring and the receiving tube in sequence, drop an absorption liquid on the separation membrane. The absorption liquid is an acid-base indicator that can produce a color change after absorbing acidic gas and the color change range is at pH = 5-9, and then install the sealing cover. Heat the sample plate, and immediately take a picture of the absorption liquid after the heating ends to obtain an image of the absorption liquid. Analyze the RGB three-primary color composition of the absorption liquid image through image recognition software to obtain the R value, G value, and B value, and construct a standard curve of G / B value - Escherichia coli concentration or (R + G) / B value - Escherichia coli concentration. Prepare a sample of the milk powder to be tested with sterile water and culture it at 37 °C to obtain a culture solution of the milk powder to be tested. Add the culture solution of the milk powder to be tested into the sample pool of the detection device according to any one of claims 1-5, place and install the separation membrane, the sealing ring and the receiving tube in sequence, drop an absorption liquid on the separation membrane, and then install the sealing cover. Heat the sample plate, and immediately take a picture of the absorption liquid after the heating ends to obtain an image of the absorption liquid. Analyze the RGB three-primary color composition of the absorbent liquid image through image recognition software to obtain the R value, G value, and B value. Substitute the G / B value into the G / B value - Escherichia coli concentration standard curve or substitute the (R + G) / B value into the (R + G) / B value - Escherichia coli concentration standard curve to obtain the Escherichia coli concentration in the milk powder sample to be tested.

8. The rapid detection method of Escherichia coli in milk powder according to claim 7, characterized in that, The heating temperature of the sample plate is 110 °C and the heating time is 15 min; or the heating temperature is 37 °C and the heating time is 24 h.

9. The rapid detection method of Escherichia coli in milk powder according to claim 7, characterized in that, The culture time of the milk powder sample to be tested and the Escherichia coli standard sample before adding to the sample cell is 15 - 24 h.

10. The rapid detection method for Escherichia coli in milk powder according to any one of claims 6 to 7, characterized in that, The preparation steps of the sterile water are as follows: Prepare a solution according to the mass ratio of porcine bile salt, phosphate buffer with a pH of 6.86, and deionized water of 1:2:200, and then seal and sterilize to obtain sterile water.

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