Color developing agent for microbial culture medium and preparation method thereof, and microbial color developing culture medium

Abstract

The present application discloses a color developer for a microbial culture medium, a preparation method thereof, and a microbial color development medium. The color developer comprises a color development substance and a carrier. The color development substance is bound to the inside and / or the surface of the carrier. The carrier is water-insoluble and hydrophilic. The color developer of the present application has good dispersibility in the microbial culture medium and is not prone to agglomeration.

Description

Technical Field

[0001] The present application relates to the technical field, and in particular to a color developer for a microbial culture medium and a preparation method thereof, and a microbial color development culture medium. Background Art

[0002] Microbial chromogenic culture medium is a new type of culture medium that uses the principle of color development by the reaction of products produced by the microorganism's own metabolism with corresponding color developers to detect microorganisms. Through specific enzyme reactions, the target microorganisms produce color changes during the culture process, thereby facilitating the rapid identification and identification of specific microorganisms. This culture medium is widely used in clinical microbiology, food microbiology, environmental microbiology and other fields. It has the following advantages: 1) High sensitivity and specificity: Different types of microorganisms contain different intracellular enzymes. When the substrate of the artificially synthesized microorganism-specific enzyme is added to the colorimetric culture medium, when the target microorganism grows, the enzyme it produces reacts with the substrate, so that the color-developing group is freed and attached to the colony, forming a colony with a unique color, which can intuitively distinguish the target microorganism from other microorganisms, greatly improving the sensitivity and specificity of the detection and reducing the subsequent complex identification steps; 2) Fast and convenient: After the sample is enriched, most colorimetric culture media can be isolated and cultured for 18-24 hours, and a preliminary identification can be made based on the color of the colony. Compared with traditional methods, it saves a lot of time and labor costs, and the results are intuitive. The results can be judged by color and are clear at a glance; 3) Wide range of applications: It can be used to detect various microorganisms, such as Escherichia coli, coliform bacteria, Staphylococcus aureus, Salmonella, Listeria, Vibrio, etc., covering a variety of sample sources such as food, water, milk, ice cream, meat products, clinical samples, etc., to meet the needs of microbial detection in different fields.

[0003] At present, the commonly used color developers are mainly indole derivatives, such as 5-bromo-4-chloro-3-indole-β-D-galactoside (X-Gal), 5-bromo-4-chloro-3-indole-β-D-glucoside (X-Glu), 5-bromo-6-chloro-3-indole octyl ester (X-Citronellol or X-Cit), 5-bromo-4-chloro-3-indole-β-D-fucoside (X-Fuc), etc. However, these color developers have poor water solubility, which makes it difficult to evenly disperse them in the culture medium. In turn, it may lead to: 1) affecting the accuracy and repeatability of microbial detection: if the concentration of the color developer in some areas is too high, it may lead to false positive results; if the concentration of the color developer in some areas is too low, the target microorganisms may not be accurately detected; 2) insoluble color developers are prone to form aggregates in the culture medium due to surface tension, van der Waals force, electrostatic adsorption, etc. These aggregates are not only difficult to disperse, but may also affect the growth environment of microorganisms, such as blocking the respiratory tract of microorganisms or affecting the delivery of nutrients.

[0004] Therefore, it is necessary to provide a color developer that can be more evenly dispersed in the culture medium. Summary of the invention

[0005] The purpose of the present application is to provide a color developer for a microbial culture medium and a preparation method thereof, and a microbial color development culture medium.

[0006] This application adopts the following technical solutions:

[0007] The first aspect of the present application discloses a color developer for a microbial culture medium, the color developer comprising a color developing substance and a carrier, the color developing substance being bound to the inside and / or the surface of the carrier, and the carrier being water-insoluble and hydrophilic. It should be noted that the color developing substance is usually insoluble in water and is prone to agglomeration in the culture medium. By binding the color developing substance to the carrier, the carrier can have good dispersibility in water because it is insoluble in water and hydrophilic; in addition, the hydrophilic surface can form a stable hydration layer to prevent further agglomeration between particles, thereby being conducive to improving the dispersibility of the color developer. Therefore, by binding the color developing substance to the inside and / or the surface of the carrier, the color developing substance can be evenly dispersed in the culture medium with the help of the properties of the carrier.

[0008] In one implementation of the present application, the color-developing substance is embedded in the carrier. It should be noted that embedding the color-developing substance in the carrier can further improve the dispersibility and reduce the occurrence of agglomeration.

[0009] In one implementation of the present application, the carrier has a hydrophilic group, thereby improving the dispersibility of the developer in water through the hydrophilic group.

[0010] In one implementation of the present application, the carrier includes at least one of hydrophilic silica, zeolite molecular sieve, and porous polystyrene microspheres. It should be noted that these substances have good biocompatibility and stability, which can help avoid the impact on microbial growth and can remain stable in the culture medium; in addition, these substances and color-developing substances have good affinity, so that the color-developing substances can be better bound to the carrier, specifically, hydrophilic silica has a high specific surface area, and its surface contains a large number of hydroxyl groups (Si-OH), which can form weak van der Waals forces and hydrogen bonds with the hydrophobic groups of 5-bromo-6-chloro-3-indoloctyl ester; zeolite molecular sieve has a regular pore structure, and the pore size is at the nanoscale. The regular pore structure enables the zeolite molecular sieve to selectively adsorb molecules of a specific size, including color-developing substances such as 5-bromo-6-chloro-3-indoloctyl ester; porous polystyrene microspheres have a rich pore structure and can also provide a large number of adsorption sites.

[0011] In one implementation of the present application, the particle size of the carrier is 1 to 100 nm. It should be noted that by making the carrier nanoparticles with a particle size of 1 to 100 nm, the carrier has a larger specific surface area, and a larger surface area means more adsorption sites, making it easier for insoluble substances (color-developing substances) to be uniformly adsorbed on the surface of the nanoparticles; in addition, a larger surface area means that the hydrophilic group has more contact with water, which can help improve the dispersibility of the color developer.

[0012] In one implementation of the present application, the particle size of the carrier is 7 to 40 nm.

[0013] In one implementation of the present application, the specific surface area of ​​the carrier is 100 to 500 m 2 / g.

[0014] In one implementation of the present application, the mass ratio of the color-developing substance to the carrier is 1:5 to 1:10.

[0015] The second aspect of the present application discloses a microbial chromogenic culture medium, which includes a basic culture medium for supporting the growth of microorganisms and a color developer, and the color developer is the color developer disclosed in the first aspect of the present application.

[0016] The third aspect of the present application discloses a method for preparing a developer, comprising: dissolving a developer substance in an organic solvent to obtain a first solution; mixing the first solution with a carrier; and volatilizing the organic solvent to obtain microcapsules, namely the developer.

[0017] In one implementation of the present application, in the step of mixing the first solution and the carrier, the stirring rate is 100 to 600 rpm.

[0018] In one implementation of the present application, in the step of mixing the first solution and the carrier, ultrasonic treatment is used, the frequency of the ultrasound is 20 to 100 kHz, and the time of the ultrasonic treatment is 4 to 15 minutes.

[0019] In one implementation of the present application, the organic solvent is volatilized by vacuum drying, and the vacuum drying includes: drying at 20 to 35° C. for 2 to 6 hours.

[0020] The beneficial effects of this application are:

[0021] The color developer of the present application has good dispersibility in the microbial culture medium and is not prone to agglomeration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a diagram of the chromogenic culture medium of the examples and comparative examples of the present application.

[0023] Figure 2 It is a result diagram of microbial culture using the chromogenic culture medium of the examples and comparative examples of the present application. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood according to the description in the specification and the general technical knowledge in the art.

[0025] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0026] Microbial chromogenic culture medium has important application value in the field of microbial detection. It can quickly and intuitively identify target microorganisms through color development reactions. With the continuous development of biotechnology, the requirements for the accuracy, efficiency and convenience of microbial detection are getting higher and higher. However, the preparation of microbial chromogenic culture medium currently faces some technical challenges, one of which is the solubility and dispersibility of the chromogenic substrate. In traditional chromogenic culture medium preparation methods, the chromogenic substrate is often an insoluble substance, which makes it difficult to evenly disperse in the culture medium during the preparation process, affecting the quality of the culture medium and the detection effect. In order to solve this problem, researchers have been seeking innovative technologies and methods.

[0027] The chromogenic substrate of the microbial chromogenic culture medium is insoluble in water and is very difficult to disperse during the preparation process, which can lead to the following problems: 1) The chromogenic substrate is unevenly distributed in the culture medium, affecting the accuracy and repeatability of microbial detection. If the concentration of the chromogenic substrate in some areas is too high, it may lead to false positive results; if the concentration of the chromogenic substrate in some areas is too low, the target microorganism may not be accurately detected. 2) Insoluble chromogenic substrates are prone to form aggregates, which are not only difficult to disperse, but may also affect the growth environment of microorganisms, such as blocking the respiratory tract of microorganisms or affecting the delivery of nutrients. 3) Due to the uneven dispersion of the chromogenic substrate, when using the culture medium for microbial culture and detection, the effect of the color reaction is unstable, making it difficult to accurately determine the type and quantity of microorganisms.

[0028] In view of this, the present application creatively proposes a color developer for microbial culture medium, which carries the color developer by selecting a carrier with a nanoparticle size. The carrier has good biocompatibility, stability and affinity with the color developer, and the carrier is insoluble in water and has good hydrophilicity. Therefore, the problem that the color developer is insoluble in water and easily aggregates in the solution can be overcome by the properties of the carrier. The present application has the following advantages:

[0029] 1) Significantly improve the dispersion uniformity of the chromogenic substrate: By embedding the chromogenic substrate in nanoparticles, the dispersion problem caused by its insolubility is effectively solved, so that the chromogenic substrate can be evenly distributed in the culture medium, ensuring that during the microbial culture and detection process, each area can undergo an accurate color reaction with the target microorganism, greatly improving the accuracy and reliability of the detection.

[0030] 2) Improve the growth environment of microorganisms: After the nanoparticles are embedded in the chromogenic substrate, the aggregation of large particles of the chromogenic substrate is avoided, the physical obstacles to the growth of microorganisms are reduced, and a more uniform and suitable growth environment is provided for the microorganisms, which is conducive to the normal growth and reproduction of microorganisms and improves the cultivation efficiency of microorganisms.

[0031] 3) Enhance the stability and quality consistency of the culture medium: The microbial culture medium prepared by this technical solution has higher stability, and the color substrate is not easy to precipitate and aggregate, which ensures the stability of the performance of the culture medium during long-term storage and use. At the same time, due to the improvement of dispersion uniformity, the quality of the culture medium prepared in different batches is more consistent, reducing the risk of quality fluctuations.

[0032] 4) Expanded selection range of chromogenic substrates: For those insoluble chromogenic substrates that are difficult to apply by traditional methods, this technical solution provides an effective solution, greatly expanding the selection range of chromogenic substrates in microbial chromogenic culture media, and creating conditions for the development of new, high-performance microbial culture media.

[0033] 5) Improved efficiency and accuracy of microbial detection: Evenly dispersed chromogenic substrates can react with target microorganisms more quickly, shortening the detection time. At the same time, accurate color development makes the test results clearer and easier to judge, improving the accuracy and efficiency of microbial detection and providing strong support for rapid detection and accurate diagnosis in related fields.

[0034] The present application relates to a color developer for a microbial culture medium and a preparation method thereof, and a microbial color development culture medium.

[0035] In a specific embodiment, the color developing agent includes a color developing substance and a carrier. The color developing substance is configured to react with the metabolites of the microorganisms and develop color.

[0036] In one embodiment, the color-developing substance is hydrophobic.

[0037] In a specific embodiment, the color-developing substance is water-insoluble. Alternatively, the color-developing substance has poor water solubility, for example, a solubility lower than 20 mg / L.

[0038] In a specific embodiment, the color-developing substance includes but is not limited to 5-bromo-4-chloro-3-indole-β-D-galactoside, 5-bromo-4-chloro-3-indole-β-D-glucoside, 5-bromo-6-chloro-3-indole octyl ester, and 5-bromo-4-chloro-3-indole-β-D-fucoside.

[0039] In one specific embodiment, the carrier is configured to carry a color-developing substance.

[0040] In a specific embodiment, the color-developing substance is bound to the inside and / or the surface of the carrier. Preferably, the color-developing substance is bound to the inside of the carrier. Alternatively, the color-developing substance is embedded in the inside of the carrier. It should be noted that the color-developing substance molecules are extremely small, insoluble in water, easy to agglomerate, and have poor dispersibility. By carrying the color-developing substance on a larger carrier, the properties of the carrier can be used to improve the agglomeration and dispersibility problems.

[0041] In one embodiment, the carrier is water-insoluble. Alternatively, the carrier has poor water solubility, for example, a solubility of less than 20 mg / L.

[0042] In one embodiment, the carrier is hydrophilic.

[0043] In one embodiment, the carrier has a hydrophilic group, for example, a hydroxyl group, an amino group, a carboxyl group, a sulfonic acid group, an amide group, an ester group, etc.

[0044] In one embodiment, the carrier is a porous material.

[0045] In a specific embodiment, the carrier includes at least one of hydrophilic silica, zeolite molecular sieve, and porous polystyrene microspheres.

[0046] In a specific embodiment, the particle size of the carrier is 1 to 100 nm. For example, the particle size of the carrier can be 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm. Preferably, the particle size of the carrier is 7 to 40 nm.

[0047] In a specific embodiment, the specific surface area of ​​the carrier is 100 to 500 m 2 / g. For example, the specific surface area of ​​the carrier is 100m 2 / g, 200m 2 / g、300m 2 / g, 400m 2 / g、500m 2 / g.

[0048] In a specific embodiment, the density of the carrier is 0.6 to 2.6 g / m 3 For example, the density of the carrier can be 0.6 g / m 3 , 1g / m 3 , 1.5g / m 3 , 2g / m 3 or 2.6 g / m 3 Preferably, the density of the carrier is 1.09 to 2.0 g / m 3 It should be noted that it is beneficial to make the color developer have better suspension properties and disperse more evenly in the culture medium to avoid floating on the surface of the culture medium or settling at the bottom of the culture medium.

[0049] In a specific embodiment, the mass ratio of the color-developing substance to the carrier is 1:5 to 1:10. For example, the mass ratio of the color-developing substance to the carrier can be 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0050] In a specific embodiment, the microbial color development medium includes a color developer and a basic medium. The basic medium is used to support the growth of microorganisms, and its components may include a carbon source, a nitrogen source, an inorganic salt, agar, a pH indicator, a non-target microbial growth inhibitor, etc., and the specific components and contents may not be limited.

[0051] In a specific embodiment, the microorganisms include but are not limited to Escherichia coli, coliform bacteria, Staphylococcus aureus, Salmonella, Listeria, and Vibrio.

[0052] In a specific embodiment, the content of the color-developing substance in the microbial color-developing culture medium is 0.01-0.5 g / L.

[0053] In a specific embodiment, the content of the color developing agent in the microbial color developing medium is 1-10 g / L.

[0054] In a specific embodiment, the preparation method of the developer may include:

[0055] (1) dissolving a color-developing substance in an organic solvent to obtain a first solution;

[0056] (2) mixing the first solution and the carrier;

[0057] (3) The organic solvent is then evaporated to obtain microcapsules, i.e., the color developer.

[0058] In one embodiment, the organic solvent is configured to dissolve the color-developing substance.

[0059] In a specific embodiment, the organic solvent may be dimethyl sulfoxide, chloroform, ethanol, acetone, or the like.

[0060] In a specific embodiment, the organic solvent is ethanol, which can dissolve the color-developing substance well and is volatile, so it is easy to dry later.

[0061] In one specific embodiment, in step (1), the color-developing substance is completely dissolved in the organic solvent.

[0062] In a specific embodiment, in step (1), the mass volume ratio of the color-developing substance to the organic solvent is 0.1 to 1 g / 100 mL.

[0063] In one specific embodiment, through step (2) and step (3), the color-developing substance is embedded in the carrier.

[0064] In a specific embodiment, in step (2), the steps include: stirring at a rate of 100 to 600 rpm; using ultrasonic treatment, the ultrasonic frequency of which is 20 to 100 kHz, and the ultrasonic treatment time of which is 4 to 15 min.

[0065] In a specific embodiment, in step (3), the organic solvent is volatilized by vacuum drying, and the vacuum drying includes: drying at 20 to 35° C. for 2 to 6 hours.

[0066] The present application is further described in detail below by specific examples. The following examples are only further described for the present application and should not be construed as limiting the present application. In the present example, unless otherwise specified, the reagents and instruments used are all commercially available, and the experimental operations are carried out in accordance with the product instructions and conventional experimental specifications.

[0067] Example:

[0068] Materials preparation:

[0069] Carrier: Hydrophilic fumed silica, nanoparticle size 7-40nm, specific surface area 380m 2 / g.

[0070] Color-developing substance: 5-bromo-6-chloro-3-indoloctyl ester, and the solvent is anhydrous ethanol.

[0071] Basic culture medium formula: tryptone 9.1g / L, beef extract powder 3g / L, glucose 3g / L, sodium chloride 3g / L, agar 15g / L, sodium deoxycholate 1.5g / L, pH adjuster: dipotassium hydrogen phosphate 0.4g / L; selection agent: cefuroxime sodium 0.04g / L.

[0072] Embedding treatment:

[0073] 1) 3 g of hydrophilic fumed silica was calcined and activated in a dry atmosphere; the calcination temperature was 200 degrees Celsius, the calcination atmosphere was high-purity nitrogen, and the calcination time was 6 hours.

[0074] 2) Dissolve 0.3 g of the insoluble chromogenic substrate 5-bromo-6-chloro-3-indoloctyl ester in 50 mL of anhydrous ethanol, stir well, and cool in a refrigerator at 4°C for later use;

[0075] 3) adding the activated silicon dioxide in step 1) to the solution prepared in step (2), stirring vigorously at a speed of 200 rpm for 15 min, and ultrasonically dispersing for 15 min at a frequency of 20 kHz. In this step, the temperature is controlled not to exceed 30° C.;

[0076] 4) Put the liquid in step 3) into a vacuum drying oven, and slowly dry it (vacuum drying for 3 hours) at a temperature of 30° C. to evaporate the solvent and form microcapsules for later use.

[0077] Medium preparation:

[0078] 1) Mix the embedded microcapsules and inhibitors by ball milling at a speed of 240 rpm for 15 min and at a temperature not exceeding 30°C;

[0079] 2) Add 0.4 g of dipotassium hydrogen phosphate, 1.5 g of sodium deoxycholate, 3 g of beef extract powder, 3 g of glucose, and 3 g of sodium chloride, and continue ball milling at a speed of 240 rpm for 15 min. The ball milling temperature should not exceed 30° C.

[0080] (3) Continue to add 9.1 g of tryptone and 15 g of agar, and mill at a speed of 240 rpm for 15 min. The milling temperature should not exceed 30°C, and then add water to 1 L and stir evenly.

[0081] Comparative Example: The color-developing substance was not embedded in a carrier, that is, 0.3 g of 5-bromo-6-chloro-3-indoloctyl ester was directly added to the basic culture medium as a color-developing agent, and the rest was the same as in the example.

[0082] Figure 1 It is a picture of the chromogenic culture medium of the embodiment and the comparative example of the present application. The right side is the chromogenic culture medium of the embodiment, and the left side is the chromogenic culture medium of the comparative example. It can be seen that the chromogenic culture medium of the embodiment has higher transparency and better dispersion effect of the chromogenic substance, while the chromogenic culture medium of the comparative example is relatively turbid, has lower transparency, and poor dispersion effect of the chromogenic substance.

[0083] 1 mL of the bacterial suspension of Salmonella typhimurium (100 CFU) was inoculated into the chromogenic medium of the embodiment and the chromogenic medium of the comparative example, and cultured at room temperature. Figure 2 This is a result diagram of microbial culture using the chromogenic culture medium of the present application example and the comparative example. Figure 2 In the figure, the two culture dishes on the right are cultured using the color development medium of the embodiment, and the two culture dishes on the left are cultured using the color development medium of the comparative example. It can be seen that the color development is faster and more obvious when the color development medium of the embodiment is used.

[0084] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A color developer for a microbial culture medium, characterized in that: The developer comprises a color developing substance and a carrier, wherein the color developing substance is combined with the inside and / or the surface of the carrier, and the carrier is water-insoluble and hydrophilic.

2. The developer according to claim 1, characterized in that The color-developing substance is embedded in the carrier.

3. The developer according to claim 1, characterized in that The carrier has a hydrophilic group; Preferably, the carrier includes at least one of hydrophilic silica, zeolite molecular sieve, and porous polystyrene microspheres.

4. The developer according to any one of claims 1 to 3, characterized in that The particle size of the carrier is 1 to 100 nm; Preferably, the particle size of the carrier is 7 to 40 nm; Preferably, the specific surface area of ​​the carrier is 100 to 500 m 2 / g.

5. The developer according to claim 1, characterized in that The mass ratio of the color-developing substance to the carrier is 1:5 to 1:

10.

6. A microbial chromogenic culture medium, characterized in that: The microbial color development medium comprises a basic medium for supporting the growth of microorganisms and a color developer, and the color developer is the color developer according to any one of claims 1 to 5.

7. A method for preparing a developer according to any one of claims 1 to 5, characterized in that: include: Dissolving a color-developing substance in an organic solvent to obtain a first solution; mixing the first solution and a carrier; The organic solvent is volatilized to obtain microcapsules, namely the developer.

8. The preparation method according to claim 7, characterized in that: In the step of mixing the first solution and the carrier, the stirring rate is 100 to 600 rpm.

9. The preparation method according to claim 8, characterized in that: In the step of mixing the first solution and the carrier, ultrasonic treatment is adopted, the frequency of the ultrasound is 20 to 100 kHz, and the time of the ultrasonic treatment is 4 to 15 minutes.

10. The preparation method according to claim 7, characterized in that: The organic solvent is volatilized by vacuum drying, wherein the vacuum drying comprises: drying at 20 to 35° C. for 2 to 6 hours.

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