A method for rapid detection of microorganisms in beverages

Abstract

The present invention relates to the field of microbial detection technology, and in particular to a method for rapid detection of microorganisms in beverages. The method comprises placing a beverage sample under a constant temperature of 30-35°C for 24 hours, and then adding it to a culture bottle with a sensing hydrogel and culturing it for 2 days for colorimetric detection; wherein the sensing hydrogel is composed of Schiff base ZIF-90, neutral red, sodium alginate and calcium chloride, which can absorb carbon dioxide produced by microbial respiration, thereby undergoing a significant color change from yellow to red. The advantage of the present invention is that the detection time is significantly shortened, and by simplifying the operating process and reducing equipment requirements, a more economical and efficient detection is achieved, which is particularly suitable for microbial safety monitoring in the food and beverage industry to ensure product quality and consumer safety.

Description

Technical Field

[0001] The invention relates to the technical field of microorganism detection, and in particular to a method for rapidly detecting microorganisms in beverages. Background Art

[0002] In the process of beverage production and quality control, it is crucial to ensure the microbial safety of products. However, there are many problems with existing microbial detection technologies, which limit their efficiency and accuracy in practical applications. Although traditional plate count and membrane filtration methods can provide accurate results, the detection time is long, and it usually takes several days to get the results. In addition, these methods are complicated to operate and require strict aseptic operation and professional technicians to operate, which not only increases labor costs but also increases the risk of errors. In addition, traditional methods may not be sensitive enough when detecting low concentrations of microorganisms, and it is difficult to distinguish different types of microorganisms.

[0003] Rapid detection technologies such as immunoassays and nucleic acid amplification techniques, although they can provide faster results, have high false positive and false negative rates in complex matrices (such as beverages), mainly due to matrix interference and insufficient specificity of the detection system. In addition, these highly sensitive rapid detection technologies usually require expensive instruments and reagents, which may bring a high economic burden to small and medium-sized enterprises. The complexity of sample processing is also a problem. Many rapid detection technologies require complex sample pretreatment steps to remove interfering substances and concentrate target microorganisms, which not only increases the number of operation steps, but may also introduce errors and variations. Therefore, there is an urgent need to further develop and optimize new detection methods to improve detection efficiency and accuracy, reduce costs, and simplify the operation process, so as to better meet the needs of the food and beverage industry for microbial detection and ensure product quality and safety. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method for rapidly detecting microorganisms in beverages.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention proposes a method for rapid detection of microorganisms in beverages, which specifically comprises the following steps:

[0006] S1. Place the test beverage sample in a constant temperature incubator at 30-35°C for 24 hours and set it as the sample to be tested;

[0007] S2. Visually inspect the culture bottles and culture dishes before starting the test to prevent the use of culture bottles and culture dishes that show signs of damage, leakage or deterioration. Disinfect the outer surface of the culture bottles and culture dishes with 75% alcohol and place them in a sterile room under ultraviolet light for 30 minutes;

[0008] S3, put the sensing hydrogel into the bottom of the culture bottle sterilized in step S2, with a thickness of 1-2 cm, and place the culture dish 2-3 cm above the sensing hydrogel, leaving a gap next to it;

[0009] S4. Before using the culture bottle, wipe the bottle mouth with a disposable sterile cotton swab, and then wipe off the residual moisture with a dry disposable cotton swab. Use a disposable syringe to draw 5 mL of the sample to be tested prepared in step S1 and inject it into the culture dish, mark it as the test bottle, and set up a blank control bottle at the same time;

[0010] S5. Place the test bottle and blank control bottle treated in step S4 into an incubator, set the incubation temperature to 30°C, and control the ambient temperature to 25°C. After 2 days of incubation, observe the color of the sensing hydrogel. If the sensing hydrogel changes from yellow to red, it means that microorganisms are present; if it remains yellow, it means that no microorganisms are present. The content of microorganisms is determined based on the degree of color change.

[0011] Preferably, the method for preparing the sensing hydrogel comprises the following steps:

[0012] A1. Dissolve imidazole-2-carboxaldehyde, zinc nitrate and sodium formate in methanol by ultrasonic dispersion, transfer to a reactor and heat at 120°C for 16-24 h, cool to room temperature and centrifuge to obtain a precipitate, wash with ethanol and deionized water alternately three times and then vacuum dry to obtain ZIF-90;

[0013] A2, adding ZIF-90 obtained in step A1 into methanol, adding anthranilic acid after uniform dispersion, heating and reflux at 60-80°C for 12-24 h, cooling to room temperature and centrifuging to obtain a precipitate, washing with ethanol and deionized water alternately three times and vacuum drying to obtain Schiff base ZIF-90;

[0014] A3. Mix the Schiff base ZIF-90 obtained in step A2 with neutral red in distilled water, adjust the pH to 10 after mixing evenly, then add sodium alginate, continue to add calcium chloride after mixing evenly, and form a sensing hydrogel after cross-linking and curing.

[0015] Preferably, in step A1, the usage ratio of imidazole-2-carboxaldehyde, zinc nitrate, sodium formate and methanol is 2-4 mmol: 1 mmol: 1-2 mol: 40 mL.

[0016] Preferably, in step A2, the ratio of ZIF-90, methanol and anthranilic acid is 50 mg: 6-10 mL: 1 mmol.

[0017] Preferably, in step A3, the dosage ratio of Schiff base ZIF-90, neutral red and distilled water is 50 mg: 10-30 mg: 50 mL, and the dosage ratio of sodium alginate, calcium chloride and distilled water is 1-1.5 g: 1-2 g: 50 mL.

[0018] The beneficial effects achieved by the present invention are as follows:

[0019] The present invention significantly shortens the detection time, simplifies the operation process, and reduces the detection cost through an innovative method for rapid detection of microorganisms in beverages. The sensing hydrogel material plays a key role in the method. The sensing hydrogel can adsorb carbon dioxide and react with it to change color, and the presence of microorganisms is quickly indicated by this color change reaction. Microorganisms produce carbon dioxide when breathing in a culture dish. Since the mass of carbon dioxide is greater than that of air, it will gather in the sensing hydrogel at the bottom of the bottle. The introduction of Schiff base ZIF-90 further improves the adsorption capacity of hydrogel for carbon dioxide, making the color change of neutral red and carbon dioxide more obvious, which is convenient for observing and judging the test results. Compared with the traditional plate counting method and membrane filtration method, this method shortens the detection time and greatly improves the detection efficiency. In addition, the hydrogel not only fixes the sensing solution, but also has a good carbon dioxide adsorption capacity. Based on the color change principle of pH change caused by the reaction of carbon dioxide and water, the accuracy of the test results is ensured. The material innovation of the sensing hydrogel makes it perform well in capturing and adsorbing carbon dioxide. The efficient adsorption performance of Schiff base ZIF-90 enhances the sensitivity and accuracy of detection, and the obvious contrast between neutral red and carbon dioxide reaction color change more intuitively indicates the presence of microorganisms. This innovative material and method is not only economical and affordable, but also has high detection efficiency and accuracy, which meets the high requirements of the food and beverage industry for microbial detection and ensures the quality and safety of the product. In short, the present invention has significant advantages in improving detection efficiency, simplifying operating procedures, reducing costs, and improving sensitivity and accuracy, and can better meet the needs of the food and beverage industry for microbial detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a schematic diagram of the structure of the culture bottle, culture dish and sensing hydrogel proposed by the present invention;

[0022] Figure 2It is the UV-visible absorption spectrum of neutral red solution under alkaline conditions and after passing CO2;

[0023] Figure 3 is a TEM image of the Schiff base ZIF-90 prepared in the process of Example 1;

[0024] Figure 4 is a SEM image of the sensing hydrogel prepared in Example 1;

[0025] Figure 5 is a microorganism concentration-grayscale fitting diagram obtained by the method described in Example 1;

[0026] Figure 6 This is a microbial concentration-grayscale fitting diagram obtained by the method described in Comparative Example 1;

[0027] Figure 7 This is a microbial concentration-grayscale fitting diagram obtained by the method described in Example 2.

[0028] Explanation of reference numerals: 1. culture bottle, 2. culture dish, 3. sensing hydrogel. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through examples and comparative examples, but this should not be understood as the scope of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples and comparative examples are purchased from commercial channels unless otherwise specified.

[0031] Example 1: This example proposes a method for rapid detection of microorganisms in beverages, which specifically includes the following steps:

[0032] S1. Place the test beverage sample in a constant temperature incubator at 30°C for 24 hours and set it as the sample to be tested;

[0033] S2. Visually inspect the culture bottle 1 and culture dish 2 before starting the test to prevent the use of culture bottles 1 and culture dishes 2 that are damaged, leaking or deteriorated. Disinfect the outer surface of the culture bottle 1 and culture dish 2 with 75% alcohol and place them in a sterile room under ultraviolet light for 30 minutes.

[0034] S3, put the sensing hydrogel 3 into the bottom of the culture bottle 1 sterilized in step S2, with a thickness of 2 cm, and clamp the culture dish 2 2 cm above the sensing hydrogel 3, leaving a gap next to it;

[0035] S4. Before using culture bottle 1, wipe the bottle mouth with a disposable sterile cotton swab, and then wipe off the residual moisture with a dry disposable cotton swab. Use a disposable syringe to draw 5 mL of the sample to be tested prepared in step S1 and inject it into culture dish 2, which is marked as a test bottle, and a blank control bottle is set at the same time;

[0036] S5. Place the test bottle and blank control bottle treated in step S4 into an incubator, set the incubation temperature to 30°C, and control the ambient temperature to 25°C. After 2 days of incubation, observe the color of the sensing hydrogel 3. If the sensing hydrogel 3 changes from yellow to red, it means that microorganisms are present; if it remains yellow, it means that no microorganisms are present. The content of microorganisms is determined based on the degree of color change.

[0037] The preparation method of the sensing hydrogel 3 comprises the following steps:

[0038] A1. Dissolve 4 mmol imidazole-2-carboxaldehyde, 1 mmol zinc nitrate and 1 mmol sodium formate in 40 mL methanol by ultrasonic dispersion, transfer to a reactor and heat at 120°C for 24 h, cool to room temperature and centrifuge to obtain a precipitate, wash with ethanol and deionized water alternately three times and then vacuum dry to obtain ZIF-90;

[0039] A2, add 50 mg of ZIF-90 obtained in step A1 into 10 mL of methanol, disperse evenly, add 1 mmol of anthranilic acid, heat and reflux at 80°C for 12 h, cool to room temperature and centrifuge to obtain a precipitate, wash with ethanol and deionized water alternately three times and vacuum dry to obtain Schiff base ZIF-90;

[0040] A3. Mix 50 mg of the Schiff base ZIF-90 obtained in step A2 and 20 mg of neutral red in 50 mL of distilled water. After mixing evenly, adjust the pH to 10, then add 1 g of sodium alginate, and then add 1.2 g of calcium chloride after mixing evenly. After cross-linking and curing, form a sensing hydrogel 3.

[0041] Example 2: This example proposes a method for rapid detection of microorganisms in beverages, which specifically includes the following steps:

[0042] S1. Place the test beverage sample in an open state in a constant temperature incubator at 35°C for 24 hours and set it as the sample to be tested;

[0043] S2. Visually inspect the culture bottle 1 and culture dish 2 before starting the test to prevent the use of culture bottles 1 and culture dishes 2 that are damaged, leaking or deteriorated. Disinfect the outer surface of the culture bottle 1 and culture dish 2 with 75% alcohol and place them in a sterile room under ultraviolet light for 30 minutes.

[0044] S3, put the sensing hydrogel 3 into the bottom of the culture bottle 1 sterilized in step S2, with a thickness of 1 cm, and clamp the culture dish 2 3 cm above the sensing hydrogel 3, leaving a gap next to it;

[0045] S4. Before using culture bottle 1, wipe the bottle mouth with a disposable sterile cotton swab, and then wipe off the residual moisture with a dry disposable cotton swab. Use a disposable syringe to draw 5 mL of the sample to be tested prepared in step S1 and inject it into culture dish 2, which is marked as a test bottle, and a blank control bottle is set at the same time;

[0046] S5. Place the test bottle and blank control bottle treated in step S4 into an incubator, set the incubation temperature to 30°C, and control the ambient temperature to 25°C. After 2 days of incubation, observe the color of the sensing hydrogel 3. If the sensing hydrogel 3 changes from yellow to red, it means that microorganisms are present; if it remains yellow, it means that no microorganisms are present. The content of microorganisms is determined based on the degree of color change.

[0047] The preparation method of the sensing hydrogel 3 comprises the following steps:

[0048] A1. Dissolve 2 mmol imidazole-2-carboxaldehyde, 1 mmol zinc nitrate and 1 mmol sodium formate in 40 mL methanol by ultrasonic dispersion, transfer to a reactor and heat at 120 °C for 16 h, cool to room temperature and centrifuge to obtain a precipitate, wash with ethanol and deionized water alternately three times and vacuum dry to obtain ZIF-90;

[0049] A2. Add 50 mg of ZIF-90 obtained in step A1 into 6 mL of methanol, disperse evenly, add 1 mmol of anthranilic acid, heat and reflux at 60°C for 24 h, cool to room temperature and centrifuge to obtain a precipitate, wash three times with ethanol and deionized water alternately, and then vacuum dry to obtain Schiff base ZIF-90;

[0050] A3. Mix 50 mg of the Schiff base ZIF-90 obtained in step A2 and 10 mg of neutral red in 50 mL of distilled water. After mixing evenly, adjust the pH to 10, then add 1.5 g of sodium alginate, and then add 2 g of calcium chloride after mixing evenly. After cross-linking and curing, form a sensing hydrogel 3.

[0051] Example 3: This example proposes a method for rapid detection of microorganisms in beverages, which specifically includes the following steps:

[0052] S1. Place the test beverage sample in a constant temperature incubator at 32°C for 24 hours and set it as the sample to be tested;

[0053] S2. Visually inspect the culture bottle 1 and culture dish 2 before starting the test to prevent the use of culture bottles 1 and culture dishes 2 that are damaged, leaking or deteriorated. Disinfect the outer surface of the culture bottle 1 and culture dish 2 with 75% alcohol and place them in a sterile room under ultraviolet light for 30 minutes.

[0054] S3, put the sensing hydrogel 3 into the bottom of the culture bottle 1 sterilized in step S2, with a thickness of 2 cm, and clamp the culture dish 2 3 cm above the sensing hydrogel 3, leaving a gap next to it;

[0055] S4. Before using culture bottle 1, wipe the bottle mouth with a disposable sterile cotton swab, and then wipe off the residual moisture with a dry disposable cotton swab. Use a disposable syringe to draw 5 mL of the sample to be tested prepared in step S1 and inject it into culture dish 2, which is marked as a test bottle, and a blank control bottle is set at the same time;

[0056] S5. Place the test bottle and blank control bottle treated in step S4 into an incubator, set the incubation temperature to 30°C, and control the ambient temperature to 25°C. After 2 days of incubation, observe the color of the sensing hydrogel 3. If the sensing hydrogel 3 changes from yellow to red, it means that microorganisms are present; if it remains yellow, it means that no microorganisms are present. The content of microorganisms is determined based on the degree of color change.

[0057] The preparation method of the sensing hydrogel 3 comprises the following steps:

[0058] A1. Dissolve 3 mmol imidazole-2-carboxaldehyde, 1 mmol zinc nitrate and 2 mmol sodium formate in 40 mL methanol by ultrasonic dispersion, transfer to a reactor and heat at 120°C for 18 h, cool to room temperature and centrifuge to obtain a precipitate, wash with ethanol and deionized water alternately three times and vacuum dry to obtain ZIF-90;

[0059] A2, add 50 mg of ZIF-90 obtained in step A1 into 8 mL of methanol, disperse evenly, add 1 mmol of anthranilic acid, heat and reflux at 70°C for 12 h, cool to room temperature and centrifuge to obtain a precipitate, wash with ethanol and deionized water alternately three times and vacuum dry to obtain Schiff base ZIF-90;

[0060] A3. Mix 50 mg of the Schiff base ZIF-90 obtained in step A2 and 30 mg of neutral red in 50 mL of distilled water. After mixing evenly, adjust the pH to 10, then add 1.2 g of sodium alginate, and then add 1 g of calcium chloride after mixing evenly. After cross-linking and curing, form a sensing hydrogel 3.

[0061] Comparative Example 1: This comparative example proposes a method for rapid detection of microorganisms in beverages. The only difference between the comparative example and Example 1 is that the Schiff base ZIF-90 is not added, and the remaining components, component contents, and experimental steps are the same as those in Example 1.

[0062] Comparative Example 1: This comparative example proposes a method for rapid detection of microorganisms in beverages. The only difference between this comparative example and Example 1 is that sodium alginate and calcium chloride are not added, and the remaining components, component contents, and experimental steps are the same as those in Example 1.

[0063] Experimental Example 1: The UV-visible absorption spectra of neutral red solution and after CO2 was introduced at pH 10 were measured using a UV-visible spectrophotometer.

[0064] Figure 2 This is the UV-visible absorption spectrum of neutral red solution under alkaline conditions and after the introduction of CO2. As shown in the figure, a new absorption peak appeared at 533 nm after the introduction of CO2 into the solution, and the solution changed from yellow to red, indicating that neutral red reacted with CO2 in water.

[0065] Experimental Example 2: The surface morphology of the Schiff base ZIF-90 prepared in Example 1 was observed using a projection electron microscope, and the microscopic morphology of the sensing hydrogel 3 prepared in Example 1 was observed using a scanning electron microscope.

[0066] Figure 3 This is a TEM image of the Schiff base ZIF-90 prepared in the process of Example 1. As shown in the figure, the Schiff base ZIF-90 still maintains a cubic morphology and the structure is not destroyed. Figure 4 3 is a SEM image of the sensing hydrogel 3 prepared in Example 1. As shown in the figure, Schiff base ZIF-90 is embedded in the three-dimensional cross-linked structure of the hydrogel, indicating that the sensing hydrogel 3 of the present invention is successfully prepared.

[0067] Experimental Example 3: Mobile phone-assisted standard colorimetric quantitative detection

[0068] Escherichia coli samples of different concentrations were prepared and cultured according to the methods described in Example 1, Comparative Example 1 and Comparative Example 2, respectively. After the culture was completed, the color change of the sensing hydrogel 3 was observed, and photos were taken under a fixed light source using a mobile phone. Image processing was performed using ImageJ software, and the obtained RGB values ​​were converted into grayscale values, which were fitted and analyzed with the microbial concentration.

[0069] Figure 5 This is the microbial concentration-grayscale fitting diagram obtained by the method described in Example 1. As shown in the figure, the microbial concentration is 10 2 ~10 7 Within the range, gray value = 42.8 + 12.8ln (concentration + 1), R 2 =0.97, with good sensitivity and good fit. Figure 6 This is a microbial concentration-grayscale fitting diagram obtained by the method described in Example 1. As shown in the figure, when the microbial concentration is 10 2 ~107 Within the range, gray value = 47.0 + 3.76ln (concentration + 1), R 2 =0.91, both sensitivity and goodness of fit are poor. Figure 7 This is a microbial concentration-grayscale fitting diagram obtained by the method described in Example 2. As shown in the figure, when the microbial concentration is 10 2 ~10 7 Within the range, gray value = 44.0 + 7.26ln (concentration + 1), R 2 =0.92, and the sensitivity and the goodness of fit are between Example 1 and the comparative example. The above analysis shows that the introduction of Schiff base ZIF-90 and hydrogel can improve the sensitivity of neutral red to carbon dioxide, as well as the stability of detection and improve the accuracy.

[0070] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for rapid detection of microorganisms in beverages, characterized in that: The specific steps include: S1. Place the test beverage sample in an open incubator at 30-35°C for 24 hours to set it as the sample to be tested; S2. Visually inspect the culture bottles and culture dishes before starting the test to prevent the use of culture bottles and culture dishes that show signs of damage, leakage or deterioration. Disinfect the outer surface of the culture bottles and culture dishes with 75% alcohol and place them in a sterile room under ultraviolet light for 30 minutes; S3, put the sensing hydrogel into the bottom of the culture bottle sterilized in step S2, with a thickness of 1-2 cm, and place the culture dish 2-3 cm above the sensing hydrogel, leaving a gap next to it; S4. Before using the culture bottle, wipe the bottle mouth with a disposable sterile cotton swab, and then wipe off the residual moisture with a dry disposable cotton swab. Use a disposable syringe to draw 5 mL of the sample to be tested prepared in step S1 and inject it into the culture dish, mark it as the test bottle, and set up a blank control bottle at the same time; S5. Place the test bottle and the blank control bottle after the treatment in step S4 into an incubator, set the incubation temperature to 30°C, and control the ambient temperature to 25°C. After 2 days of incubation, observe the color of the sensing hydrogel. If the sensing hydrogel changes from yellow to red, it means that microorganisms are present. If it remains yellow, it means that no microorganisms are present. The content of microorganisms is determined based on the degree of color change. The preparation method of the sensing hydrogel comprises the following steps: A1. Dissolve imidazole-2-carboxaldehyde, zinc nitrate and sodium formate in methanol by ultrasonic dispersion, transfer to a reactor and heat at 120°C for 16-24 h, cool to room temperature and centrifuge to obtain a precipitate, wash with ethanol and deionized water alternately three times and then vacuum dry to obtain ZIF-90; A2, adding ZIF-90 obtained in step A1 into methanol, adding anthranilic acid after uniform dispersion, heating and reflux at 60-80°C for 12-24 h, cooling to room temperature and centrifuging to obtain a precipitate, washing with ethanol and deionized water alternately three times and vacuum drying to obtain Schiff base ZIF-90; A3, the Schiff base ZIF-90 obtained in step A2 and neutral red are mixed in distilled water, the pH is adjusted to 10 after mixing evenly, and then sodium alginate is added, and calcium chloride is added after mixing evenly, and the sensing hydrogel is formed after cross-linking and curing; In the step A1, the ratio of imidazole-2-carboxaldehyde, zinc nitrate, sodium formate and methanol is 2-4 mmol: 1 mmol: 1-2 mol: 40 mL; The ratio of ZIF-90, methanol and anthranilic acid in step A2 is 50 mg: 6-10 mL: 1 mmol; In the step A3, the dosage ratio of Schiff base ZIF-90, neutral red and distilled water is 50 mg: 10-30 mg: 50 mL, and the dosage ratio of sodium alginate, calcium chloride and distilled water is 1-1.5 g: 1-2 g: 50 mL.

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