A colorimetric diluent for food microbiology determination and its preparation method

Abstract

This invention discloses a colorimetric diluent for the determination of microorganisms in food and its preparation method. The colorimetric diluent comprises sterile physiological saline and Brilliant Blue FCF at a final concentration of 0.0001%~0.1%. By adding a trace amount of food-grade blue pigment to the diluent, a clear blue color is achieved, allowing operators to visually judge the mixing state of the sample and diluent through the uniformity of the blue color, thus visualizing the series of dilution processes. This invention also discloses the preparation method of the colorimetric diluent and its application in the determination of total bacterial count. Experimental verification shows that the colorimetric diluent has no significant effect on the growth of common food microorganisms, does not affect the coagulation performance of PCA medium, and is fully compatible with the testing procedures of the national standard GB 4789.2-2022. This invention is low-cost, easy to operate, and can effectively reduce operator variability, making it particularly suitable for teaching and training, grassroots quality inspection training, and microbiological testing in standardized laboratories, with good prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a colorimetric diluent for food microbial determination and its preparation method, which is particularly suitable for the sample dilution step in the determination of total bacterial count in food microbial testing, as well as for visual auxiliary operation in teaching experiments and standardized testing training. Background Technology

[0002] Microbiological testing of food is a crucial step in ensuring food safety. According to national standards such as GB 4789.2-2022 (National Food Safety Standard - Microbiological Examination of Food: Determination of Total Colony Count) and GB 4789.15-2016 (National Food Safety Standard - Microbiological Examination of Food: Count of Molds and Yeasts), food microbiological testing requires mixing the sample with a diluent (such as sterile physiological saline or phosphate buffer) in a specific ratio, performing a series of serial dilutions, and then inoculating the diluted solution into a culture medium for incubation and colony counting. The mixing effect in the dilution step directly affects the accuracy and repeatability of subsequent test results.

[0003] Currently, in standardized operations and teaching experiments for food microbiology testing, the mixing effect of the dilution step relies entirely on the operator's experience and sense of responsibility, lacking intuitive and objective visual aids. Operators typically judge whether the mixture is homogeneous by visually observing the appearance of the diluent or by feel. When sample particles are small and the diluent is colorless and transparent, it is difficult to accurately determine whether the sample has been uniformly dispersed. This is especially true in serial dilution processes, where operational errors are easily amplified at each stage, ultimately leading to inaccurate colony counts. This problem is particularly prominent in batch sample testing, teaching practice, and grassroots quality inspection training, seriously affecting the accuracy of test results and the standardization of experimental teaching.

[0004] To address the issue of judging the mixing effect during sample dilution, some existing technologies have attempted to improve the performance of the diluent by adding functional components. For example, invention CN110964775A discloses a method for detecting the number of Bacillus subtilis, which uses physiological saline containing an emulsifier (Tween 20 or Tween 60) to dilute the test sample. The emulsifier improves the surface tension of Bacillus subtilis in physiological saline, promoting uniform dispersion and thus improving detection accuracy. Although this technical solution addresses the issue of dispersion uniformity in the diluent, it solves the problem of uneven distribution caused by Bacillus subtilis floating due to its light weight. The technical means used is to add surfactants to change the physical dispersion properties, aiming to make the bacteria evenly distributed in the diluent, rather than providing the operator with a direct basis for judging the mixing effect.

[0005] Furthermore, invention CN110804644A discloses a method and apparatus for detecting multiple microorganisms in food. This method adds chromogenic compounds (bromothymol blue and xylenol blue) to the culture medium and determines the presence of target microorganisms in the petri dish through a colorimetric reaction. Invention CN111206066A discloses a rapid detection method for microorganisms, which uses a coliform test strip containing a chromogenic indicator for detection and counts colonies through a colorimetric reaction on the test strip. Both of the above patent documents involve the use of chromogenic agents in the "culture stage" or "detection stage" for the identification or counting of microorganisms. The stage and purpose of the chromogenic agents in these patents are significantly different from those of this invention, and neither involves the technical concept of adding chromogenic agents to the diluent to visualize the dilution process.

[0006] Therefore, current technologies still lack an auxiliary method that can intuitively and conveniently assess the mixing effect during the dilution step, is fully compatible with national standard testing procedures, and does not affect microbial growth and counting. Especially for teaching experiments and standardized operation training scenarios, there is an urgent need for a low-cost, easy-to-use, and equipment-free visual dilution solution to improve the accuracy and repeatability of experimental operations. Summary of the Invention

[0007] This invention addresses the technological gap in existing food microbiology testing methods, where dilution steps rely entirely on operator experience to judge mixing effectiveness and lack intuitive visualization aids. It provides a colorimetric diluent for food microbiology determination and its preparation method. This invention aims to solve the technical problem in existing technologies where the colorless and transparent nature of the diluent makes it difficult to visually assess sample dispersion, leading to amplified operational errors during serial dilution processes. This is particularly problematic in teaching, practical training, and grassroots quality inspection training, affecting the accuracy and repeatability of test results.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A colorimetric diluent for the determination of microorganisms in food comprises sterile physiological saline and brilliant blue FCF as a visual indicator; the final concentration of brilliant blue FCF in the colorimetric diluent is 0.0001%~0.1% (w / v).

[0009] Furthermore, the purity of the Brilliant Blue FCF is ≥85%, and the Brilliant Blue FCF complies with the national food safety standard GB 2760-2014 and the requirements of FDA 21 CFR 74.340, ensuring its safety and regulatory compliance in food microbiological testing.

[0010] Furthermore, the sterile physiological saline is a sodium chloride aqueous solution with a mass-volume ratio of 0.85%~0.90%, which meets the requirements of Appendix A.3 of GB 4789.2-2022, and the colorimetric diluent does not affect the solidification performance of the subsequent plate counting agar (PCA) medium after being used for sample dilution.

[0011] Furthermore, the final concentration of the brilliant blue FCF in the colorimetric diluent is 0.001%~0.01% (w / v), so as to form a continuous color gradient that can be seen by the naked eye during the tenfold serial dilution process, and to achieve intuitive distinction between different dilution factors.

[0012] The present invention also provides a method for preparing the above-mentioned colorimetric diluent for food microbiological determination, comprising the following steps: Step 1: Dissolve Brilliant Blue FCF in a portion of sterile physiological saline to prepare a Brilliant Blue FCF stock solution with a mass-to-volume ratio of 0.1% to 10%. Step 2: Mix the Brilliant Blue FCF stock solution with the remaining sterile saline solution and dilute to a final Brilliant Blue FCF concentration of 0.0001%~0.1% (w / v). Step 3: The mixture obtained in Step 2 is filtered and sterilized to obtain the colorimetric diluent for food microbiology determination.

[0013] Furthermore, in step three, the filtration and sterilization are performed using a filter membrane with a pore size of 0.22 μm to ensure the sterility of the diluted solution.

[0014] The present invention also provides the application of the above-mentioned colorimetric diluent in the determination of microorganisms in food, the application including using the colorimetric diluent in a series of dilution steps of food samples before the determination of total bacterial count, and judging the mixing effect by observing the uniformity of the blue color of the diluent.

[0015] Furthermore, the total bacterial count is determined in accordance with the national food safety standard GB 4789.2-2022; the colorimetric diluent replaces conventional sterile saline in the dilution step without changing the detection process and culture conditions, thus achieving seamless integration with the current national standard.

[0016] Furthermore, the colorimetric diluent is used in teaching experiments or standardized testing training. The color gradient formed by the series of ten-fold dilutions visually demonstrates the dilution factor, helps to judge the accuracy of pipetting and mixing operations, and significantly reduces operational errors.

[0017] The present invention also provides a colorimetric dilution reagent kit for the determination of microorganisms in food, the kit comprising: individually packaged colorimetric dilution, or brilliant blue FCF stock solution and sterile physiological saline for preparing the colorimetric dilution; and an instruction manual, the instruction manual describing the operation guidelines for judging the mixing status by the color uniformity of the colorimetric dilution in the determination of total bacterial count.

[0018] Compared with the prior art, the present invention has the following outstanding advantages: (1) This invention adds 0.0001%~0.1% Brilliant Blue FCF to sterile physiological saline to make the diluent a clear blue color. Operators can visually judge the mixing state of the sample and the diluent by the uniformity of the blue color, thus realizing the visualization of the series of dilution processes. The results of test example 3 show that after using the colorimetric diluent of this invention, the overall coefficient of variation of colony count results among different experienced operators decreased from 13.6% to 5.1%, which significantly reduced the impact of differences in operator experience. The verification of example 3 and test example 1 shows that Brilliant Blue FCF in this concentration range has no significant effect on the growth of common food microorganisms such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger, and does not affect the coagulation performance of PCA medium (example 2). It is fully compatible with the national standard test procedure of GB 4789.2-2022. Test example 4 shows that the relative deviation from the test results of the national standard method is within ±10%.

[0019] (2) This invention is low in cost, easy to operate, requires no additional equipment, and is ready to use immediately. Comparative Examples 1 and 2 show that colorless physiological saline in the prior art lacks visualization assistance, while diluents containing emulsifiers, although improving dispersibility, cannot provide intuitive judgment and may affect colony morphology. Example 5 further provides a kit form, which is convenient for direct use in laboratories and teaching institutions. Test Example 2 shows that the colorimetric diluent of this invention has good color stability after 60 days of storage at 4°C, making it suitable for commercial production and application. The above results indicate that this invention is particularly suitable for teaching and training, grassroots quality inspection training, and microbial detection in standardized laboratories, and has good prospects for industrial application. Attached Figure Description

[0020] Figure 1The image shows the color gradient of the physiological saline solution containing brilliant blue FCF in a series of ten-fold dilutions provided in Example 1 of this invention. The concentrations of brilliant blue FCF in tube 1# are 1.0% (10 mg / mL), 0.1% (1.0 mg / mL), 0.01% (0.1 mg / mL), 0.001% (0.01 mg / mL), 0.001% (0.01 mg / mL), and 0.0001% (0.001 mg / mL) in tube 5#. Tube 6 is a control containing physiological saline solution without brilliant blue FCF. Figure 2 The image shows the mixing effect of the brilliant blue diluent and PCA culture medium provided in Example 2 of this invention; the left image shows the state before mixing, and the right image shows the state after thorough mixing. Figure 3 The following is a diagram showing the results of culturing Escherichia coli DH5α at the same concentration in PCA medium with / without brilliant blue, as provided in Example 3 of this invention; wherein plate (1) is the chromogenic dilution group containing 1.0% brilliant blue FCF, plate (2) is the chromogenic dilution group containing 0.1% brilliant blue FCF, and plate (3) is the physiological saline control group without brilliant blue FCF. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 Preparation of colorimetric diluent and observation of color gradient.

[0023] This embodiment provides a colorimetric diluent for the determination of microorganisms in food, and its preparation method is as follows: (1) Preparation of mother liquor: Accurately weigh 1.0 g of brilliant blue FCF (purity ≥85%, conforming to GB 2760-2014 standard) and dissolve it in 100 mL of sterile physiological saline (0.85% NaCl, w / v). Stir magnetically until completely dissolved to obtain a brilliant blue FCF mother liquor with a mass-volume ratio of 1.0%.

[0024] (2) Filtration and sterilization: The above mother liquor was filtered and sterilized using a 0.22 μm sterile filter membrane. The filtrate was collected in a sterile container and stored at 4°C in the dark for later use.

[0025] (3) Preparation of working solution: Take the above mother solution and dilute it in sterile physiological saline according to the ratio to prepare colorimetric dilution solutions with final concentrations of Brilliant Blue FCF of 1.0%, 0.1%, 0.01%, 0.001%, and 0.0001% respectively (i.e., 10 mg / mL, 1.0 mg / mL, 0.1 mg / mL, 0.01 mg / mL, and 0.001 mg / mL).

[0026] The different concentrations of the colorimetric diluents were placed into test tubes numbered 1# to 6#, respectively. Test tube 1# contained 1.0% (10 mg / mL) Brilliant Blue, test tube 2# contained 0.1% (1.0 mg / mL) Brilliant Blue, test tube 3# contained 0.01% (0.1 mg / mL) Brilliant Blue, test tube 4# contained 0.001% (0.01 mg / mL) Brilliant Blue, test tube 5# contained 0.0001% (0.001 mg / mL) Brilliant Blue, and test tube 6# contained physiological saline diluent without Brilliant Blue FCF as a control.

[0027] Results observation: such as Figure 1 As shown, test tubes 1# to 5# exhibit a continuous color gradient from dark blue to light blue. The blue color gradually lightens with increasing dilution factor, allowing the operator to visually distinguish between different dilution factors. Test tube 6# is colorless and transparent. These results demonstrate that the chromogenic diluent of this invention can form a clearly discernible color gradient within a concentration range of 0.0001% to 1.0%, providing visual assistance for a series of dilution operations.

[0028] Example 2 Compatibility verification of the chromogenic diluent with PCA medium.

[0029] This embodiment verifies the effect of the colorimetric diluent on the solidification properties of plate counting agar (PCA) medium.

[0030] Take 1 mL of the Brilliant Blue FCF final concentration of 0.1 mg / mL (0.01%) prepared in Example 1, add it to 15-20 mL of PCA medium that has been melted and cooled to about 45°C, mix gently, pour into a sterile Petri dish, and let it stand at room temperature to solidify.

[0031] Results observation: such as Figure 2 As shown, (left) the image shows the state before mixing, where the culture medium and the chromogenic diluent are not completely mixed, and the blue color is unevenly distributed; (right) the image shows the state after thorough mixing, where the blue color is evenly distributed in the culture medium, and the surface of the petri dish is smooth after solidification, without cracks or liquefaction. This result indicates that the brilliant blue FCF in the chromogenic diluent of this invention, at a concentration of 0.01%, does not affect the solidification performance of PCA culture medium, is no different from that of conventional physiological saline diluent, and is fully compatible with subsequent pour-over culture procedures.

[0032] Example 3 Verification of the effect of colorimetric dilution on microbial growth.

[0033] This embodiment verifies the effect of different concentrations of brilliant blue FCF chromogenic dilution on the growth of Escherichia coli DH5α.

[0034] (1) Preparation of bacterial suspension: Take fresh Escherichia coli DH5α culture and dilute it appropriately with sterile physiological saline to prepare a bacterial suspension with a suitable dilution (bacterial concentration of about 10^4~10^5 CFU / mL).

[0035] (2) Group settings: Experimental group 1: A colorimetric diluent containing 1.0% (10 mg / mL) Brilliant Blue FCF was used as the diluent; Experimental Group 2: A colorimetric diluent containing 0.1% (1.0 mg / mL) Brilliant Blue FCF was used as the diluent; Control group: Sterile saline without brilliant blue FCF was used as the diluent.

[0036] (3) Inoculation and culture: Take 1 mL of each of the three dilutions and mix them evenly with 9 mL of bacterial suspension. Then, take 1 mL of the mixture and pour it into a 90 mm diameter petri dish. Add 15-20 mL of sterilized PCA medium and gently rotate the petri dish to mix evenly. After the agar solidifies, invert the dish and incubate it in a 37℃ constant temperature incubator for 48 h.

[0037] (4) Colony counting: After the culture is completed, the colonies in each petri dish are counted.

[0038] Results observation: such as Figure 3 As shown, plate (1) is experimental group 1 (1.0% Brilliant Blue concentration), with 45 colonies; plate (2) is experimental group 2 (0.1% Brilliant Blue concentration), with 44 colonies; plate (3) is the control group (without Brilliant Blue), with 46 colonies. The number of colonies in the three groups is similar, and there are no significant differences in colony morphology and size.

[0039] The results indicate that within the Brilliant Blue FCF concentration range (0.0001%~0.1%) defined in this invention, the chromogenic dilution has no significant inhibitory or promoting effect on the growth of Escherichia coli and does not affect the accuracy of colony counting results.

[0040] Example 4 Application of colorimetric diluent in the dilution of food samples.

[0041] This embodiment verifies the application effect of the colorimetric diluent of the present invention in the dilution process of an actual food sample (chicken mince).

[0042] (1) Sample preparation: Weigh 25 g of minced chicken sample, place it in a sterile homogenizing bag, add 225 mL of color development diluent containing Brilliant Blue FCF with a final concentration of 0.01% (0.1 mg / mL), and homogenize with a tapping homogenizer for 1~2 min to obtain a 1:10 sample homogenate.

[0043] (2) Serial dilution: Using a 1 mL sterile pipette, take 1 mL of the 1:10 sample homogenate and inject it into a test tube containing 9 mL of colorimetric diluent (0.01% Brilliant Blue FCF concentration). Shake well to obtain a 1:100 sample homogenate. Repeat the above operation to prepare a series of dilutions of 1:1000, 1:10000, and 1:100000.

[0044] (3) Judgment of mixing effect: During the series of dilutions, the operator judges the mixing effect by observing the uniformity of the blue color in the test tube. When the shaking is sufficient, the blue color in the entire test tube is uniform; when the shaking is insufficient, uneven color or layering can be seen in some areas.

[0045] Results: In this embodiment, the operator can immediately adjust the shaking intensity and time by visually judging the uniformity of the blue color, ensuring that the sample homogenate of each dilution gradient is fully mixed and avoiding sampling errors caused by uneven mixing. Simultaneously, the test tubes of different dilution gradients exhibit a continuous color gradient from dark blue to light blue, making it easy for the operator to confirm the current dilution factor and effectively preventing pipetting errors.

[0046] Example 5 Preparation of the colorimetric dilution reagent kit.

[0047] This embodiment provides a colorimetric dilution reagent kit for the determination of microorganisms in food.

[0048] (1) Component A: Brilliant Blue FCF concentrate stock solution. Accurately weigh 5.0 g of Brilliant Blue FCF (purity ≥85%), dissolve it in 100 mL of sterile physiological saline, filter it through a 0.22 μm filter membrane for sterilization, dispense it into sterile vials, 10 mL per vial, and seal the vials.

[0049] (2) Component B: Sterile physiological saline. Prepare a 0.85% NaCl aqueous solution, dispense it into sterile bottles, 90 mL per bottle, and autoclave at 121℃ for 15 min.

[0050] (3) Instructions for use: The instructions explain in detail how to mix component A and component B before use and shake well to obtain a Brilliant Blue FCF final concentration of 0.05% colorimetric diluent, which can be directly used for a series of dilution operations on food samples. The instructions also describe the key points of operation for judging the mixing state by the uniformity of blue color.

[0051] This kit can be transported at room temperature (avoiding high temperatures) and can be mixed before use, making it convenient for laboratories and teaching institutions to use immediately without the need for preparation and sterilization.

[0052] Comparative Example 1 Diluted with colorless physiological saline (existing technology comparison).

[0053] In accordance with the provisions of GB 4789.2-2022, sterile physiological saline (0.85% NaCl) without any colorimetric components was used as the diluent, and a series of dilution operations were performed on 25 g of chicken mince sample in the same manner as in Example 4.

[0054] Three operators with 1-3 years of experience in microbial testing independently performed the dilution procedure and visually assessed the mixing effect after dilution. Results showed that because the diluent was colorless and transparent, operators found it difficult to accurately determine whether the sample was fully mixed, especially at high dilution ratios (1:10000 and above), where sample particles were no longer visible to the naked eye. The operators had to rely entirely on their experience to judge the shaking time and force. Two of the three operators exhibited varying degrees of uneven mixing, resulting in poor parallelism in subsequent colony counting results (relative standard deviation RSD > 15%).

[0055] This comparative example shows that the colorless saline solution used in the existing technology lacks visual aids, the mixing effect depends on the operator's experience, and is prone to operational errors, affecting the accuracy and repeatability of the test results.

[0056] Comparative Example 2 Dilute using a diluent containing emulsifier (refer to CN110964775A).

[0057] Referring to the technical solution of the prior art document CN110964775A, physiological saline containing Tween 20 (1% by volume) was prepared as a diluent, and a series of dilution operations were performed on 25 g of chicken mince sample according to the same method as in Example 4.

[0058] During the operation, because Tween 20 is a colorless surfactant, the diluted solution remained colorless and transparent, making it impossible for the operator to visually assess the mixing effect, a problem similar to that of Comparative Example 1. Furthermore, after pouring the diluted solution into PCA medium, some Petri dishes exhibited diffuse colony growth with blurred colony boundaries, making counting difficult. Analysis suggests that Tween 20, as a surfactant, may have altered the surface tension of the culture medium, affecting the normal growth morphology of the colonies.

[0059] This comparative example shows that although the use of emulsifiers can improve the dispersibility of bacteria in the dilution, it cannot provide a visual judgment of the mixing effect and may have an adverse effect on subsequent culture, making it unsuitable for routine colony count determination.

[0060] Comparative Example 3 The concentration of Brilliant Blue FCF is too high (beyond the scope of this invention).

[0061] Prepare a chromogenic dilution of Brilliant Blue FCF with a final concentration of 5.0% (50 mg / mL), and conduct microbial growth effect experiments in the same manner as in Example 3.

[0062] The results showed that the number of *E. coli* DH5α colonies cultured in this high-concentration chromogenic dilution was only 12, far lower than the 46 colonies in the control group, and the colonies were smaller and grew slowly. This indicates that when the concentration of Brilliant Blue FCF exceeds 0.1%, it significantly inhibits microbial growth and affects the accuracy of colony counting results.

[0063] This comparative example verifies the necessity and criticality of the brilliant blue FCF concentration range (0.0001%~0.1%) defined in this invention; beyond this range, it cannot be guaranteed that microbial growth will not be affected.

[0064] Test Example 1 Effects of Brilliant Blue FCF on the growth of different microorganisms To verify the compatibility of the colorimetric diluent of this invention with the growth of different types of food microorganisms, five common food microorganisms were selected for testing.

[0065] Test strain: Escherichia coli ( Escherichia coli ATCC 25922, Staphylococcus aureus ( Staphylococcus aureus ATCC 6538, Bacillus subtilis ( Bacillus subtilis ATCC 6633, brewer's yeast ( Saccharomyces cerevisiae ATCC 9763, Aspergillus niger ( Aspergillus niger ATCC16404.

[0066] Test method: Fresh cultures of each strain were prepared into bacterial suspensions of appropriate concentrations using sterile physiological saline. These suspensions were then serially diluted 10-fold using a chromogenic diluent containing 0.01% Brilliant Blue FCF and sterile physiological saline (blank control). The appropriate dilutions were then used for plate pour culture. Colony counts were performed after incubation. Each strain was tested in triplicate.

[0067] Test results: The results are shown in Table 1.

[0068] Results analysis: The colony counts of the above five strains in the chromogenic dilution containing 0.01% Brilliant Blue FCF showed no significant difference compared with the blank control group, with relative deviations within ±6%. This result indicates that the chromogenic dilution of the present invention, at a concentration of 0.01%, has no significant effect on the growth of common food microorganisms such as bacteria, yeasts, and molds, demonstrating good broad-spectrum compatibility.

[0069] Test Example 2 Color stability test of colorimetric diluent To verify the color stability of the chromogenic diluent of the present invention under different storage conditions, the chromogenic diluent with a final concentration of 0.01% Brilliant Blue FCF was dispensed into sterile test tubes and stored in the dark at 4℃, 25℃, and 37℃ respectively. Samples were taken at 0 days, 7 days, 14 days, 30 days, and 60 days respectively, and the absorbance value at a wavelength of 630 nm was measured using a UV-Vis spectrophotometer, and the color change was observed.

[0070] Test results: The results are shown in Table 2.

[0071] Results analysis: After 60 days of storage at 4℃, the absorbance of the colorimetric diluent remained essentially unchanged, with no significant color change. At 25℃, the color remained stable for 30 days, but the absorbance decreased by approximately 2.5% after 60 days. At 37℃, the color decay was more pronounced, with the absorbance decreasing by approximately 7.7% after 60 days. These results indicate that the colorimetric diluent of this invention exhibits good color stability under 4℃ refrigeration conditions and can maintain its colorimetric effect for a relatively long period, making it suitable for industrial production and commercial applications.

[0072] Test Example 3 Consistency test of dilution and mixing effect among different operators To verify the effectiveness of the chromogenic diluent of this invention in reducing inter-operator variability, six operators with different experience in microbial testing (experience years: 0.5 years, 1 year, 2 years, 3 years, 5 years, and 8 years) were selected in our unit. They used the chromogenic diluent of this invention (Brilliant Blue FCF 0.01%) and conventional colorless physiological saline to perform a series of dilutions on the same batch of minced chicken samples. After dilution to 1:10000, the total bacterial count of each dilution was determined. Three replicates were set up for each operator, and the coefficient of variation (CV%) of the bacterial count results among different operators was compared.

[0073] Test results: The results are shown in Table 3.

[0074] Results Analysis: When using conventional physiological saline, there were significant differences in colony counts among operators with varying experience, with an overall coefficient of variation of 13.6%. This was particularly pronounced among novice operators with less than 0.5 years of experience, where the coefficient of variation reached as high as 28.6%. However, using the chromogenic diluent of this invention allowed operators to visually assess the mixing effect using the uniformity of the blue color, significantly reducing the impact of operator experience differences. The overall coefficient of variation decreased to 5.1%, and even the coefficient of variation for novice operators decreased to 8.2%. These results demonstrate that the chromogenic diluent of this invention can effectively reduce inter-operator variability, improve the consistency and reproducibility of test results, and is particularly suitable for teaching, training, and applications in grassroots laboratories.

[0075] Test Example 4 Conformity verification of the colorimetric diluent with the national standard method In accordance with the provisions of GB 4789.2-2022, commercially available food samples (three batches each of cooked meat products, pasteurized milk, and frozen dumplings) were selected. The total bacterial count of the same batch of samples was determined using the colorimetric diluent of this invention (Brilliant Blue FCF 0.01%) and the sterile physiological saline specified in the national standard. Three replicates were set up for each sample to compare the differences in the detection results of the two methods.

[0076] Test results: The results are shown in Table 4.

[0077] Results Analysis: The total bacterial count results obtained using the chromogenic diluent of this invention were highly consistent with those obtained using the national standard method, with relative deviations within ±10%, showing no significant difference. This result indicates that the chromogenic diluent of this invention fully complies with the testing requirements of GB 4789.2-2022 and can be used as a direct substitute for conventional physiological saline without requiring any modifications to existing testing procedures.

[0078] In summary, the colorimetric diluent for food microbial assays and its preparation method provided by this invention achieve visualization of the dilution process by adding a trace amount of brilliant blue FCF to sterile physiological saline. Operators can intuitively judge the mixing effect through the uniformity of the blue color, significantly reducing operational errors. It is particularly suitable for teaching, practical training, and grassroots quality inspection training. This colorimetric diluent has no effect on the growth of common food microorganisms, is fully compatible with national standard testing procedures, is low in cost, and is easy to operate, showing good prospects for industrial application.

Claims

1. A colorimetric diluent for the determination of microorganisms in food, characterized in that, The chromogenic diluent contains sterile physiological saline and brilliant blue FCF as a visual indicator; the final concentration of brilliant blue FCF in the chromogenic diluent is 0.0001%~0.1% (w / v).

2. The colorimetric diluent for food microbiological determination according to claim 1, characterized in that, The purity of the Brilliant Blue FCF is ≥85%, and the Brilliant Blue FCF complies with the requirements of GB 2760-2014 National Food Safety Standard and FDA 21 CFR74.

340.

3. The colorimetric diluent for food microbiological determination according to claim 1, characterized in that, The sterile physiological saline is a sodium chloride aqueous solution with a mass-volume ratio of 0.85% to 0.90%, and the colorimetric diluent does not affect the solidification performance of the subsequent plate counting agar (PCA) medium after being used to dilute the sample.

4. The colorimetric diluent for food microbiological determination according to claim 1, characterized in that, The final concentration of the brilliant blue FCF in the colorimetric diluent is 0.001%~0.01% (w / v) to form a continuous color gradient that is visible to the naked eye during a tenfold serial dilution.

5. A method for preparing a colorimetric diluent for food microbiological determination as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Dissolve Brilliant Blue FCF in a portion of sterile physiological saline to prepare a Brilliant Blue FCF stock solution with a mass-to-volume ratio of 0.1% to 10%. Step 2: Mix the Brilliant Blue FCF stock solution with the remaining sterile saline solution and dilute to a final Brilliant Blue FCF concentration of 0.0001%~0.1% (w / v). Step 3: The mixture obtained in Step 2 is filtered and sterilized to obtain the colorimetric diluent for food microbiology determination.

6. The preparation method according to claim 5, characterized in that, In step three, filtration and sterilization are performed using a filter membrane with a pore size of 0.22 μm.

7. The application of the colorimetric diluent as described in any one of claims 1-4 in the determination of microorganisms in food, characterized in that, The application includes using the colorimetric diluent in a series of dilution steps of food samples before total bacterial count determination, and judging the mixing effect by observing the uniformity of the blue color of the diluent.

8. The application according to claim 7, characterized in that, The total bacterial count was determined in accordance with the national food safety standard GB 4789.2-2022; the colorimetric diluent replaced the conventional sterile saline in the dilution step without changing the detection process or culture conditions.

9. The application according to claim 7, characterized in that, The colorimetric diluent is used in teaching experiments or standardized testing training. The color gradient formed by the series of ten-fold dilutions visually demonstrates the dilution factor, helping to judge the accuracy of pipetting and mixing operations.

10. A colorimetric dilution reagent kit for the determination of microorganisms in food, characterized in that, The kit includes: The colorimetric diluent according to any one of claims 1-4, individually packaged, or the brilliant blue FCF stock solution and sterile physiological saline used to prepare the colorimetric diluent according to any one of claims 1-4; And the instruction manual, which describes the operational guidelines for judging the mixing status by the color uniformity of the colorimetric diluent in the determination of total bacterial count.

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