A method for high-sensitivity rapid detection of colony total number by integrating short-time culture and signal sensitization technology

Abstract

The present application belongs to the technical field of food detection, and particularly relates to a method for high-sensitivity rapid detection of colony total number by integrating short-time culture and signal sensitization technology. The method comprises the following steps: sample pretreatment based on buffer solution and LB liquid medium, lateral flow test strip enrichment and capture, in-situ color development by EDTA-2Na-MTT, and result interpretation. The method couples short-time culture and signal sensitization to construct a triple signal amplification path, relies on the synergistic amplification of color development signal by the regulation of active bacteria proliferation and bacterial outer membrane permeability, can greatly shorten the detection period, and is far superior to the traditional 48 h plate counting method. Meanwhile, the method only responds to active live bacteria, can reduce false positives, and the pretreatment can eliminate the interference of matrixes such as pigments, high salt, starch and protein, and the detection limit is as low as 100 CFU / mL. The method does not require large instruments, supports visual and grayscale dual interpretation, has excellent performance, and is suitable for on-site rapid detection of various complex food matrices.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a highly sensitive and rapid method for detecting total bacterial count by integrating short-time culture and signal enhancement technology. Background Technology

[0002] Total bacterial count is a core microbiological indicator for evaluating food hygiene quality, monitoring the production process, and releasing products. Its detection efficiency directly affects the timeliness and accuracy of food safety supervision. For example, soy sauce, as a traditional fermented condiment, frequently exceeds the total bacterial count limit, which can cause irreparable and significant losses to both businesses and consumers. According to GB 2717-2018, the National Food Safety Standard for Soy Sauce, the limit for total bacterial count in soy sauce is ≤3×10⁻⁶. 4 However, most companies limit their internal control standards to well below 3 × 10 CFU / mL to ensure safe product release. 4 CFU / mL. As an important grain raw material, the total bacterial count on the surface of wheat grains is a key indicator for assessing the hygienic quality of the raw material. However, current traditional methods for detecting total bacterial count require specialized laboratories and personnel, resulting in long testing cycles. Although various rapid detection methods for total bacterial count have been developed domestically and internationally to address these issues, they struggle to simultaneously meet the requirements of rapid on-site detection and high sensitivity. Furthermore, rapid detection technologies for total bacterial count on complex matrices such as soy sauce and wheat grains remain relatively scarce. Therefore, establishing a rapid, accurate, and highly sensitive method for detecting total bacterial count is of great significance for the quality control of soy sauce and wheat.

[0003] Currently, the main method for detecting total bacterial count in food is the plate count method specified in GB 4789.2, "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count". The basic procedure of this method includes: sample dilution, pouring into petri dishes, incubation at 36℃±1℃ for 48±2 h, and counting report. Although the plate count method is considered the "gold standard" for total bacterial count detection and has the advantages of accuracy and high authority, it has the following inherent drawbacks: (i) The testing cycle is too long. The plate count method requires an incubation time of 48±2 hours, and it usually takes 2 to 3 days from sample collection to the issuance of the test report. For food production enterprises, the long testing cycle will result in products not being sold in a timely manner, leading to higher storage costs and storage contamination risks.

[0004] (ii) The operation is cumbersome and requires specialized instruments and personnel. The traditional plate count method involves a cumbersome sample processing and testing process. In order to avoid environmental pollution, it usually needs to be carried out in a laboratory equipped with specialized instruments. Human error caused by different operators can also significantly interfere with the accuracy and stability of the test results.

[0005] (III) Interference from complex food matrices in detection. For samples with complex matrices, general rapid detection methods are subject to significant color and component interference. For example, there are currently no corresponding standards for sample pretreatment methods and total bacterial count detection methods for wheat grains. Moreover, wheat grains have high starch and protein content. When using the traditional plate count method to detect total bacterial count after grinding, the starch particles and total bacterial count are mixed and difficult to distinguish with the naked eye.

[0006] To address the aforementioned issues, researchers have developed various rapid detection technologies for total bacterial count in recent years, such as instrumental analysis, ATP bioluminescence assays, and immunological techniques. The basic operational procedures for these methods include sample pretreatment, incubation or signal acquisition, instrument interpretation, and result output. While rapid detection methods, as a supplement to plate counting, offer advantages such as short detection time, ease of operation, and the ability to perform on-site screening, they still have the following limitations: (i) Insufficient accuracy in counting "live bacteria". Most rapid detection methods (such as ATP bioluminescence assay) cannot effectively distinguish the metabolic signals of live and dead bacteria. The detection results are usually estimated based on the total adenosine triphosphate (ATP) content, which is easily affected by free ATP and residual ATP from dead bacteria in the sample, leading to higher detection values ​​and thus misjudging the actual total number of live bacteria. When repeated detection of the sample is required, the signal attenuation may be severe due to changes in enzyme substrate activity, resulting in poor repeatability and stability of the detection.

[0007] (II) Limited sensitivity and resistance to matrix interference. Currently, the detection limits of rapid detection methods are generally higher than those of traditional plate counting methods, resulting in insufficient detection capability for low-concentration contaminated samples and a high likelihood of false negatives. Furthermore, the complex composition of food matrices, such as pigments in soy sauce and starch and protein components in wheat grains, can inhibit enzyme reactions or interfere with photoelectric signal acquisition, leading to significant errors in detection results. In addition, some rapid detection methods have stringent requirements for sample pretreatment; improper pretreatment can further amplify the matrix effect, failing to effectively reflect the true microbial contamination status of the sample and thus failing to meet the release requirements of enterprises.

[0008] In summary, developing a rapid detection method for total bacterial count that is simple to operate, low in cost, has a short detection cycle, and is applicable to complex food matrices is of significant practical importance and has broad application prospects. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies and address issues such as long detection cycles, cumbersome operations, requirement for specialized instruments and personnel, insufficient accuracy in counting viable bacteria, and limited sensitivity and resistance to matrix interference, this invention provides a rapid method for detecting total bacterial count. This method organically couples a dual amplification strategy of "short-time culture-signal enhancement," utilizing the biochemical compatibility and synergistic enhancement effect of the signal transduction chain to construct a triple signal amplification pathway of "viable bacterial metabolic proliferation → membrane permeability regulation → in-situ colorimetric enrichment." This achieves excellent comprehensive detection performance in complex matrices, with the overall detection limit (100 CFU / mL) significantly lower than the theoretical lower limit when each individual technique is used alone. Ultimately, this enables rapid and highly sensitive detection of total bacterial count in complex matrix samples.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a highly sensitive and rapid method for detecting total bacterial count by integrating short-time incubation and signal sensitization. The method includes the following steps: S1. Sample pretreatment: After sterilization, the food sample is diluted or surface-washed with PBS buffer solution under aseptic conditions, and then LB liquid medium is added for short-term incubation of no more than 6 hours to allow the target viable bacteria in the sample to proliferate to a detectable level, while eliminating matrix interference. S2. Enrichment and capture: The pretreated test liquid is added to the sample pad of the lateral flow test strip, and the bacteria are enriched and fixed in situ on the reaction pad by means of capillary action. S3, In-situ color development: The enriched and captured lateral flow test strip is placed in MTT color development solution containing EDTA-2Na and the color development reaction is carried out for more than 30 minutes. EDTA-2Na regulates the permeability of the bacterial outer membrane and promotes the entry of MTT into the viable bacteria to carry out metabolic color development reaction. S4. Result Interpretation: The total number of bacterial colonies in the sample is obtained by visual colorimetric semi-quantitative analysis or gray value analysis.

[0011] In the detection method of this invention, the pretreatment step uses a buffer solution and LB liquid culture medium. On the one hand, appropriate dilution or elution rapidly eliminates background interference from soy sauce pigments, high salt content, and amino acids, as well as matrix interference from starch and protein macromolecules on the surface of wheat grains. On the other hand, short-term dynamic culture in the treatment tube allows the target viable bacteria to rapidly proliferate to the detectable threshold at a low initial concentration. For soy sauce and wheat grains, this invention optimizes the best short-time culture methods requiring only 6 hours and 1 hour, respectively, significantly shortening the culture time compared to the traditional 48-hour culture method.

[0012] The detection process utilizes a lateral flow test strip composed of a glass fiber membrane, a nitrocellulose membrane, and an absorbent pad. This strip achieves in-situ enrichment and immobilization of bacteria through physical retention and surface adsorption, providing a homogeneous solid-phase interface for the subsequent colorimetric reaction. The test strip also integrates sample receiving, pretreatment, target capture, and waste liquid absorption functions, supporting on-site operation.

[0013] The colorimetric interpretation step introduces an MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) colorimetric solution containing disodium ethylenediaminetetraacetate (EDTA-2Na). The key is that EDTA-2Na can not only chelate residual metal ion inhibitors in complex samples, but more importantly, it can specifically bind to and remove Ca²⁺ and Mg²⁺ on the bacterial outer membrane, releasing lipopolysaccharide to enhance outer membrane permeability. This significantly promotes the entry of MTT into the mitochondria of live bacteria and its reaction with succinate dehydrogenase to generate an insoluble blue-purple formazan product. This sensitization mechanism, together with the short-term culture at the front end, forms a positive feedback loop of "proliferation-permeability-color development": the short-term culture amplifies the absolute number of live bacteria, EDTA-2Na simultaneously amplifies the color development yield of a single bacterial cell, and the two work together to amplify the color signal response amplitude, ultimately reducing the detection limit of soy sauce samples to 100 CFU / mL (total time 7-8 hours) and wheat grain samples to 1000 CFU / mL (total time 3-4 hours).

[0014] The interpretation method supports both visual colorimetric semi-quantitative and grayscale value analysis modes, without the need for external large instruments.

[0015] The method of this invention effectively solves the problem of accurate counting of "live bacteria". By using the principle of short-term proliferation coupled with metabolic activity colorimetry, it generates signal accumulation only for live bacteria with metabolic activity, avoiding false positive interference caused by dead or damaged bacteria. At the same time, by utilizing the complexation and neutralization effect of EDTA-2Na on common metal ion inhibitors in complex matrices, it significantly enhances the ability to resist matrix interference, ensuring stable detection performance even in complex backgrounds such as high salt, high amino acid content in soy sauce, and high starch content in wheat grains.

[0016] Preferably, the food samples include soy sauce and wheat grains. The highly sensitive and rapid detection method for total bacterial count in soy sauce and wheat grains based on short-time culture and signal enhancement exhibits good performance in terms of accuracy, precision, detection limit, specificity, and robustness, and can meet the practical application needs of food production enterprises for process monitoring and rapid food safety screening.

[0017] More preferably, for soy sauce samples, the short-term culture time is 4-6 h; for wheat kernel samples, the short-term culture time is >0 h and ≤1 h.

[0018] More preferably, the detection limit for soy sauce samples is 100 CFU / mL (total detection time controlled within 7-8 h), and the detection limit for wheat kernel samples is 1000 CFU / mL (total detection time controlled within 3-4 h).

[0019] Preferably, the final concentration of EDTA-2Na in the MTT chromogenic solution is 8-10 mM. By adding EDTA-2Na to the chromogenic solution, the chromogenic signal is enhanced. EDTA-2Na specifically binds to and removes magnesium and calcium ions on the bacterial outer membrane, thereby releasing lipopolysaccharide from the outer membrane. This increases the permeability of the bacterial cell membrane, allowing the chromogenic solution to enter the cell more quickly and react with bacterial metabolites to produce a visible color signal.

[0020] Preferably, the temperature of the colorimetric reaction is room temperature to 37°C.

[0021] Preferably, step S4 establishes a semi-quantitative interpretation interval based on grayscale value analysis: for soy sauce samples, a grayscale value < 3100 corresponds to a total bacterial count < 100 CFU / mL, a grayscale value of 3100–4000 corresponds to a total bacterial count of 100–1000 CFU / mL, and a grayscale value > 4000 corresponds to a total bacterial count > 1000 CFU / mL; for wheat grain samples, a grayscale value < 1500 corresponds to a total bacterial count < 1000 CFU / mL, a grayscale value of 1500–3600 corresponds to a total bacterial count of 1000–10000 CFU / mL, and a grayscale value > 3600 corresponds to a total bacterial count > 10000 CFU / mL.

[0022] Preferably, the lateral flow test strip is assembled from a glass fiber membrane, a nitrocellulose membrane, and an absorbent pad, achieving in-situ enrichment and fixation of bacteria through physical retention and surface adsorption. The test strip integrates modules for sample receiving, sample pretreatment, target enrichment, signal collection, and waste liquid treatment, enabling rapid on-site detection and immediate result output.

[0023] More preferably, the lateral flow chromatography test strip is supported by a PVC backing, with a sample pad made of glass fiber membrane at the upstream end, a reaction pad made of nitrocellulose membrane in the middle, and an absorbent pad at the downstream end; the sample pad and the reaction pad, and the reaction pad and the absorbent pad overlap each other, and the liquid migration is driven by capillary action.

[0024] Compared with the prior art, the beneficial effects of the present invention are: This invention discloses a highly sensitive and rapid method for detecting total bacterial count by integrating short-time culture and signal enhancement technologies. The method includes sample pretreatment based on buffer solution and LB liquid medium, lateral flow test strip enrichment and capture, EDTA-2Na-MTT in-situ colorimetric development, and result interpretation. This method couples short-time culture and signal enhancement to construct a triple signal amplification pathway, utilizing the synergistic amplification of the colorimetric signal through viable bacterial proliferation and bacterial outer membrane permeability regulation. This method can significantly shorten the detection cycle, with a total detection time far superior to the traditional 48-hour plate counting method. Simultaneously, based on the principle of viable bacterial metabolic colorimetric development, this method only responds to live bacteria, effectively reducing false positive interference. Furthermore, the pretreatment can effectively eliminate matrix interference from pigments, high salt, starch, and protein in food samples, achieving a detection limit as low as 100 CFU / mL. It requires no large instruments, supports dual interpretation via visual inspection and grayscale values, and exhibits excellent accuracy, precision, and anti-interference performance, making it suitable for rapid on-site detection of various complex food matrices. Attached Figure Description

[0025] Figure 1 The principle and flowchart of sample pretreatment and rapid detection; Figure 2 The specific procedures for sample pretreatment, rapid detection, result determination and method validation are illustrated using (a) soy sauce and (b) wheat kernels as examples. Figure 3 To illustrate the effect of different short-term incubation times on the color development of samples, (a) soy sauce and (b) wheat grains were used as examples. Figure 4 The effects of different sample processing methods on the samples include (a) the change in average gray value of color development with the concentration of added EDTA; (b) the effects of three pretreatment methods—no pretreatment, 10-fold dilution, and 10-fold dilution combined with EDTA—on the signal-to-noise ratio of soy sauce samples; (c) the plate culture results of bacterial colonies on the surface of wheat grains before and after elution; and (d) the statistics of the total number of bacterial colonies on the surface of wheat grains before and after elution and the calculation of the recovery rate. Figure 5 The following are evaluation results of the accuracy of the method in Example 1 for detecting total colony count in soy sauce and wheat grains: (a) Analysis of grayscale values ​​of positive and negative results for soy sauce samples; (b) Analysis of the confusion matrix for soy sauce samples; (c) Analysis of grayscale values ​​of positive and negative results for wheat grains; (d) Analysis of the confusion matrix for wheat grains. Figure 6 The results of the evaluation of the precision of the method in Example 1 for detecting the total colony count in soy sauce and wheat kernels are shown, with (a) soy sauce and (b) wheat kernels as examples respectively; Figure 7The following are the detection limits and semi-quantitative results for the total bacterial count in soy sauce and wheat kernels as determined by the method in Example 1: (a) Detection limit for soy sauce sample; (b) Semi-quantitative range for soy sauce sample; (c) Detection limit for wheat kernels; (d) Semi-quantitative range for wheat kernels. Figure 8 The results show the specificity evaluation of the method in Example 1 for detecting total bacterial count in soy sauce and wheat grains; (a) specificity of soy sauce sample; (b) specificity of wheat grains; wherein the experimental group consisted of 10 Escherichia coli. 3 CFU / mL + Staphylococcus aureus 10 3 CFU / mL (1:1), interference group 1 consisted of 10 experimental bacteria. 3 CFU / mL + Monascus purpureus 10 4 CFU / mL, interference group 2 consisted of 10 CFU / mL experimental bacteria. 3 CFU / mL + Candida albicans 10 4 CFU / mL; Figure 9 The results show the robustness assessment of the method in Example 1 for detecting total colony count in soy sauce and wheat grains; (a) robustness of the soy sauce sample; (b) robustness of the wheat grains. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0028] Example 1: A rapid method for detecting total bacterial count integrating pretreatment, detection, and color development (taking soy sauce and wheat grains as examples) 1. Pre-processing A. The specific steps for pre-processing soy sauce are as follows: A1. Take 5 mL of soy sauce stock solution (Chubang soy sauce (premium fresh soy sauce)) and add it to a sterile centrifuge tube for high-temperature sterilization (121℃, 20min). Place the sterilized soy sauce stock solution in a biosafety cabinet and perform dilution in the biosafety cabinet. A2. Add 100 μL of sterile soy sauce concentrate to a sterile centrifuge tube, then add 200 μL of a 10% concentration... 2A standard mixture of CFU / mL Escherichia coli and Staphylococcus aureus (volume ratio 1:1) and 700 μL of PBS (0.1M, pH=7.4) buffer were added to a sterile centrifuge tube. The soy sauce was then diluted 1:10, and the mixture was vortexed for 15 s to ensure complete mixing, resulting in a bacterial concentration of 2 × 10⁻⁶. 1 Soy sauce samples at CFU / mL. Strains used: *Escherichia coli* (strain No. 10907) and *Staphylococcus aureus* (strain No. 10001), both purchased from the China Industrial Microbial Culture Collection Center (CICC).

[0029] A3, bacterial concentration is 2×10 1 1 mL of liquid culture medium (LB) was added to a soy sauce sample with CFU / mL to obtain a bacterial concentration of 1×10⁻⁶. 1 A CFU / mL soy sauce sample was placed in a 37 ℃ incubator and cultured with shaking (for a short period of no more than 6 h).

[0030] B. The specific steps for pre-treatment of wheat kernels are as follows: B1. Take 1 g of wheat grains into a sterile centrifuge tube, add 1 mL of 75% ethanol to the centrifuge tube, vortex for 15 s to ensure that the 75% ethanol is fully in contact with the surface of the wheat grains, take out the wheat grains and put them into a 37 ℃ oven, wait for 5 min to allow the ethanol on the surface of the wheat grains to be completely dried. This step is to sterilize the wheat grains. B2. Place the aseptically treated wheat grains into a sterile centrifuge tube and add 200 μL of a 10% concentration... 4 A mixture of standard Escherichia coli and Staphylococcus aureus at a concentration of CFU / mL (1:1 volume ratio) and 800 μL of sterile PBS buffer (0.1M, pH 7.4) was vortexed for 15 s to ensure thorough mixing. The eluent was then pipetted into a sterile centrifuge tube, yielding a bacterial concentration of 2 × 10⁻⁶. 3 Prepare CFU / mL wheat grain eluent for use. This step involves manually spiking the wheat grains and eluting the bacteria on their surface. B3, bacterial concentration is 2×10 3 Add 1 mL of liquid culture medium (LB) to the wheat grain eluent at CFU / mL to obtain a bacterial concentration of 1×10⁻⁶. 3 CFU / mL of wheat grain eluent was placed in a 37 ℃ incubator and cultured with shaking (for a short period of no more than 6 h).

[0031] 2. The detection steps for the lateral flow chromatography test strip (composed of a glass fiber membrane, a nitrocellulose membrane, and an absorbent pad) are as follows: (1) Add 50 μL of the sample to be tested onto the sample pad. After 5 min, add another 50 μL of the sample to the sample pad and wait for 5 min. Then, add 50 μL of sterile PBS buffer to the sample pad to rinse the sample. Finally, remove the reaction pad with tweezers and place it in the colorimetric solution; (2) After placing the color developing solution containing the reaction pad at 37 °C for 30 min, remove the reaction pad from the color developing solution and observe the color intensity of the bands. (3) The total bacterial count concentration is semi-quantitatively determined by colorimetric card or by quantitative analysis of gray value using mobile software, and the total bacterial count value is output.

[0032] The colorimetric solution has been sensitized and optimized; specifically, it is an MTT / EDTA-2Na mixed colorimetric solution, and the preparation steps are as follows: (1) Weigh disodium ethylenediaminetetraacetate (EDTA-2Na) and dissolve it in sterile deionized water to prepare a 100 mM EDTA-2Na solution. Adjust the pH to 7.4 and store at 4 °C for later use. At the same time, prepare a 1 mg / mL MTT colorimetric solution (sterilized by filtration through a 0.22 μm aqueous filter) using sterile PBS (0.1 M, pH = 7.4) buffer and store it at 4 °C for later use.

[0033] (2) Before use, mix the above MTT chromogenic solution with EDTA-2Na solution in proportion, and dilute with sterile PBS to the working concentration, so that the final concentration of EDTA-2Na is 10 mM. After thorough vortex mixing, the sensitizing chromogenic working solution is obtained. EDTA-2Na in the chromogenic solution can act on the bacterial outer membrane, promote the entry of the chromogenic substrate into the bacterial cell, and promote the metabolic reductase reaction, thereby generating an enhanced visual color signal.

[0034] The principles and processes of preprocessing and rapid detection are as follows: Figure 1 As shown. 100 μL of the sample to be tested was added to a pretreatment tube, diluted with PBS buffer, and then enriched using LB liquid medium to achieve a significant increase in bacteria. 100 μL of the test solution was then pipetted onto the sample pad of the test strip. Capillary action drove the test solution to migrate laterally from the sample pad through the NC membrane to the absorbent pad. Bacteria were enriched at the junction of the sample pad and the NC membrane, while waste liquid flowed to the absorbent pad for disposal. After the sample solution had completely passed through the NC membrane, the sample pad and absorbent pad were removed. The NC membrane was then added to a mixed chromogenic solution of EDTA-2Na and MTT for in-situ color development. EDTA-2Na in the chromogenic solution increases cell membrane permeability and accelerates the reaction between MTT and succinate dehydrogenase in cells, thus achieving highly sensitive and rapid detection of total bacterial count in complex samples.

[0035] The sample pretreatment, rapid detection, result interpretation, and method validation process is as follows: Figure 2 As shown. The pretreatment process for the soy sauce samples is as follows (…). Figure 2 a) Specifically: First, the soy sauce is sterilized, then diluted 1:10 using sterile phosphate-buffered saline (PBS). The diluted solution is added to LB liquid medium and incubated at 37°C for 6 h. Subsequently, a lateral flow test strip is used to rapidly detect the total bacterial count in the incubated sample. This method enables rapid on-site detection of soy sauce samples with a bacterial concentration of 100 CFU / mL. The pretreatment process for wheat grain samples (…) Figure 2 b) Specifically: First, wheat grains are sterilized. Then, the total number of colonies on the surface is eluted using PBS eluent. The eluent is collected and added to LB liquid medium. After incubation at 37°C for 1 h, the total number of colonies in the eluent is rapidly detected using a lateral flow test strip. This method allows for rapid on-site detection of wheat grain samples with a colony concentration of 1000 CFU / mL.

[0036] Example 2: This embodiment further examines the optimization effect of different short incubation times on the colorimetric signal of sample detection, and the results are as follows: Figure 3 As shown. For the soy sauce sample ( Figure 3 a) Five incubation time gradients were set: 0 h, 1 h, 2 h, 4 h, and 6 h. Samples at time 0 h were taken immediately after adding LB medium (without further incubation) as a baseline control. For each set time point, after incubation, the sample was removed from the incubator, and 100 μL of the cultured bacterial solution was pipetted and vertically added to the center of the sample pad on a lateral flow test strip. Three test strips were prepared in parallel for each incubation time point (i.e., three repeated tests, n=3). The NC membranes obtained at each time point were completely immersed in centrifuge tubes containing a mixed MTT / EDTA-2Na colorimetric solution and allowed to react for 30 min. After incubation, the NC membrane was removed with tweezers, and grayscale analysis was performed using ImageJ image analysis software. The average grayscale value was plotted, revealing that when the short-term incubation time was 4 to 6 hours, the grayscale value of the test strip changed significantly, and the slope of the signal intensity change curve over time was the steepest, indicating the highest signal enhancement efficiency during this period. Considering both detection sensitivity and timeliness, 6 hours was determined to be the optimal short-term incubation time for soy sauce samples with a colony concentration of 100 CFU / mL. For wheat grain samples (… Figure 3b) After elution in the sample pretreatment tube, the same gradient time incubation was carried out. The results showed that the signal enhancement rate reached its maximum in the short incubation time range of 0h to 1h, and the band color intensity after 1h incubation was sufficient to meet the requirements for visualization interpretation. Therefore, in order to make the wheat sample reach the detection limit of 1000CFU / mL, the preferred short incubation time is 1h.

[0037] Example 3: This embodiment further examines the impact of different processing methods on the detection system, and the results are as follows: Figure 4 As shown, Figure 4 (a) This paper demonstrates the effect of different concentrations of EDTA-2Na sensitizing reagent on the color development signal of soy sauce samples. To determine the optimal addition concentration of EDTA-2Na in the MTT / EDTA-2Na mixed color development solution, a concentration gradient optimization experiment was set up. First, sterile stock solution of EDTA-2Na·2H2O (100 mM) and MTT stock solution (1 mg / mL) were prepared. Then, the final concentration of EDTA-2Na was adjusted to 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM, respectively, for a total of 6 gradient levels. For each EDTA-2Na concentration gradient, 3 test strips were prepared independently (i.e., 3 repeated tests, n=3). Their gray values ​​were analyzed and plotted for comparison. The results showed that as the concentration of EDTA-2Na increased, the visual color development of the test strip gradually deepened, and the corresponding gray value showed an upward trend. Based on this, the optimal addition amount of EDTA-2Na was determined to be 10 mM. Figure 4 (b) Through comparative experiments, the contributions of the 10-fold dilution pretreatment and the EDTA-2Na sensitizing reagent to eliminating the interference of soy sauce's own color and improving the detection signal-to-noise ratio (S / N) were evaluated. Four sets of comparative experiments were designed, and each set was repeated 3 times (n=3):

[0038] Group 1 (without any treatment): Take 1 mL of soy sauce stock solution, without dilution with PBS, and add 100 μL directly to the sample pad of the test strip. After chromatography, use MTT colorimetric solution without EDTA-2Na to perform the colorimetric reaction on the NC membrane. Group 2 (10-fold dilution only): The soy sauce stock solution was diluted 10-fold with PBS. 100 μL of the diluted solution was used for chromatography. The NC membrane colorimetric solution was the same as that in Group 1 (excluding EDTA-2Na). Group 3 (Dilution + EDTA-2Na Synergistic): 100 μL of the soy sauce stock solution was diluted 10 times with PBS and then subjected to chromatography. The NC membrane colorimetric solution used was MTT colorimetric solution containing 10 mM EDTA-2Na (i.e., the complete pretreatment + detection process of Example 1).

[0039] The results show that the synergistic effect of the two can effectively improve the S / N ratio and eliminate the interference of the color of the soy sauce sample itself on the detection signal, proving that the pretreatment step of the method of the present invention has good matrix adaptability.

[0040] For wheat grain samples, artificially spiked standard bacterial solutions were directly spread onto plates (10 parallel plates, n=10). Another wheat grain sample was treated according to the pretreatment and elution procedure in Example 1, and the upper eluent was used as the recovered bacterial solution. This eluent was also spread onto plates (10 parallel plates, n=10). Both experiments were incubated upside down at 37 ℃ for 48 h before counting. The total number of colonies recovered by elution was calculated based on the plate count results. Figure 4 As shown in (c, d), the colony recovery rate is as high as 95.65%, indicating that the elution method can efficiently recover target microorganisms on the surface and has high feasibility and reliability.

[0041] Example 4: This embodiment further examines the effectiveness of the method in Example 1 in evaluating the accuracy of soy sauce and wheat grains. The experimental method is as follows: the bacterial detection solution (soy sauce or wheat grain samples with artificially added bacteria (100 CFU / mL of bacteria (1:1 standard Escherichia coli and Staphylococcus aureus)) and the blank matrix (sterile soy sauce samples without added bacteria) were respectively operated according to the detection procedure in Example 1. Both groups were tested 20 times, and the detection results were compared with the results of the standard method for total bacterial count detection.

[0042] For the soy sauce sample Figure 5 (a) shows that the measurement values ​​of the positive group and the negative group are completely separated with no overlapping areas, which intuitively demonstrates that the method of Example 1 has excellent discrimination ability between positive and negative samples. Figure 5 (b) The confusion matrix further quantifies the detection performance, with sensitivity, specificity, and accuracy all at 100%, fully demonstrating the high accuracy of the method in Example 1 in soy sauce matrix. For wheat grain samples, Figure 5 (c) scatter plot and Figure 5 The confusion matrix in (d) also shows that the positive and negative samples can be clearly distinguished, and the sensitivity, specificity and accuracy all reach 100%, further verifying that the method of Example 1 has reliable accuracy in different food matrices.

[0043] Example 5: This embodiment further examines the effectiveness of the method in Example 1 in evaluating the precision of soy sauce and wheat grains. The experimental method was as follows: soy sauce and wheat grains were tested according to the method in Example 1, at 10... 2 CFU / mL-10 5Five standard bacterial solutions were selected within the CFU / mL range for testing. Each concentration was repeated 10 times. The resulting color values ​​were analyzed and statistically plotted to assess the variability of the results. Figure 6 (a) shows that the relative standard deviation (RSD) of the results measured at five different concentration levels in the soy sauce sample was less than 15%, indicating that the method of Example 1 has good repeatability and robustness in this matrix. Figure 6 (b) Similarly, it is shown that the RSD values ​​at all concentration levels in the wheat grain samples are less than 15%, further verifying that the method of Example 1 has good precision and stability in different food matrices.

[0044] Example 6: This embodiment further examines the detection limits of the method in Example 1 in two matrices: soy sauce and wheat grains, and establishes corresponding semi-quantitative result interpretation intervals. The experimental method is as follows: Soy sauce and wheat grains were tested according to the method in Example 1. Five gradient bacterial suspensions were selected near the detection limits (approximately 100 CFU / mL for soy sauce and approximately 1000 CFU / mL for wheat grains), and 10 parallel tests were performed for each. The relative standard deviation (RSD) was calculated. The lowest concentration at which the detection result showed no significant difference in accuracy and precision from the traditional method was determined as the detection limit. The experimental results are as follows: Figure 7 As shown.

[0045] For the soy sauce sample Figure 7 (a) The results showed that after short-term culture combined with EDTA-2Na sensitization treatment, the detection limit of the total bacterial count in soy sauce by the method in Example 1 could reach 100 CFU / mL; when the sample bacterial concentration was about 50 CFU / mL, the positive detection rate could reach 80%, and the relative standard deviation (RSD) was less than 10%, indicating that the method has the potential to rapidly and sensitively detect soy sauce samples with a concentration as low as 50 CFU / mL. Figure 7 (b) The grayscale value distribution range of soy sauce samples near the detection limit is further given, based on which the following semi-quantitative interpretation criteria can be established: 1. When the gray value is <3100 and the total bacterial count in the sample is <100 CFU / mL, the company can release the product; 2. When the gray value is between 3100 and 4000, the total bacterial count concentration in the sample is 100 to 1000 CFU / mL. It is recommended that the company further confirm the result using the standard plate count method before deciding on the treatment method. 3. When the gray value is >4000, the total bacterial count in the sample is >1000 CFU / mL, and the company needs to perform subsequent processing on the product.

[0046] For wheat grain samples Figure 7(c) The results showed that the detection limit of the method in Example 1 was 1000 CFU / mL. According to... Figure 7 Based on the grayscale value range shown in (d), the following semi-quantitative interpretation criteria are established: 1. When the gray value is <1500 and the total bacterial count in the sample is <1000 CFU / mL, the company can release the product; 2. When the gray value is between 1500 and 3600, the total bacterial count concentration in the sample is between 1000 and 10000 CFU / mL. It is recommended that the company further confirm the result using the standard plate count method before deciding on the treatment method. 3. When the gray value is >3600, the total bacterial count in the sample is >10000 CFU / mL, and the company needs to further process the product.

[0047] Example 7: This embodiment further examines the detection specificity of the method in Example 1 in two matrices: soy sauce and wheat grains. The experimental method is as follows: 100 CFU / mL (soy sauce) and 10... 3 Escherichia coli and Staphylococcus aureus (mixed at a 1:1 ratio) at CFU / mL (wheat grains) were used as the experimental group. Total bacterial count was measured three times in accordance with the method described in Example 1, and the grayscale value was determined after color development. Monascus purpureus and Candida albicans were prepared into 10 CFU / mL solutions. 3 CFU / mL (soy sauce), 10 4 A CFU / mL (wheat grain) bacterial suspension was prepared. This fungal suspension was mixed with the aforementioned experimental bacteria at a 1:1 ratio. The mixed suspension of *Monascus purpureus* and the experimental bacteria was designated as interference group 1, and the mixed suspension of *Candida albicans* and the experimental bacteria was designated as interference group 2. Each concentration and each combination was tested in triplicate. An equal volume of PBS was used as a blank control, and the experiment was also repeated in triplicate. The strains used were: *Monascus purpureus* (strain number D11631) purchased from the Shanghai Microbiological Culture Collection Center (SHMCC); and *Candida albicans* (strain number 10231) purchased from the American Type Culture Collection (ATCC). Results are as follows: Figure 8 As shown.

[0048] Figure 8 The results of (a) show that in the soy sauce matrix, the gray value signals of interference group 1 and interference group 2 did not change significantly compared with the experimental group, and the relative deviation was small. This indicates that fungi such as Monascus purpureus and Candida albicans have little interference with the detection of total bacterial count using the method of Example 1, indicating that the method of Example 1 has high specificity for the detection of total bacterial count. Figure 8(b) Your results also show that the aforementioned interfering bacteria did not significantly affect the detection results in wheat grain matrix, further verifying the good specificity of the method in Example 1 for the detection of target bacteria in different food matrices.

[0049] Example 8: This embodiment further examines the robustness of the method of Example 1 in soy sauce and wheat grain matrices, focusing on evaluating the effect of reaction temperature on the visualization signal. The experimental method was as follows: when using the method of Example 1 for rapid detection of total bacterial count, colorimetric reactions were performed at 37 °C and room temperature, with 10 replicates for each.

[0050] Figure 9 The results in (a) show that there is no significant difference in the gray values ​​of the soy sauce samples measured at room temperature (25 °C) and reaction conditions of 37 °C, indicating that the detection method has good tolerance to changes in reaction temperature in the soy sauce matrix. Figure 9 The results in (b) also show that there is no significant difference in the gray value of wheat grain samples at the two reaction temperatures mentioned above, indicating that the temperature fluctuation has a weak effect on the colorimetric signal of the method in Example 1 and that the method has good robustness.

[0051] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A highly sensitive and rapid method for detecting total bacterial count by integrating short-time incubation and signal enhancement, characterized in that, Includes the following steps: S1. Sample pretreatment: After sterilization, the food sample is diluted or surface-washed with PBS buffer solution under aseptic conditions, and then LB liquid medium is added for short-term incubation of no more than 6 hours to allow the target viable bacteria in the sample to proliferate to a detectable level, while eliminating matrix interference. S2. Enrichment and capture: The pretreated test liquid is added to the sample pad of the lateral flow test strip, and the bacteria are enriched and fixed in situ on the reaction pad by means of capillary action. S3, In-situ color development: The enriched and captured lateral flow test strip is placed in MTT color development solution containing EDTA-2Na and the color development reaction is carried out for more than 30 minutes. EDTA-2Na regulates the permeability of the bacterial outer membrane and promotes the entry of MTT into the viable bacteria to carry out metabolic color development reaction. S4. Result Interpretation: The total number of bacterial colonies in the sample is obtained by visual colorimetric semi-quantitative analysis or gray value analysis.

2. The highly sensitive and rapid detection method for total bacterial count integrating short-time culture and signal enhancement as described in claim 1, characterized in that, The food samples included soy sauce and wheat kernels.

3. The highly sensitive and rapid detection method for total bacterial count integrating short-time culture and signal enhancement according to claim 2, characterized in that, For soy sauce samples, the short-term incubation time was 4-6 h; for wheat kernel samples, the short-term incubation time was >0 h and ≤1 h.

4. The highly sensitive and rapid detection method for total bacterial count integrating short-time culture and signal enhancement according to claim 2, characterized in that, The detection limit for soy sauce samples was 100 CFU / mL, and the detection limit for wheat kernel samples was 1000 CFU / mL.

5. The highly sensitive and rapid detection method for total bacterial count integrating short-time culture and signal enhancement according to claim 1, characterized in that, The final concentration of EDTA-2Na in the MTT colorimetric solution is 8-10 mM.

6. The highly sensitive and rapid detection method for total bacterial count integrating short-time culture and signal enhancement according to claim 1, characterized in that, The temperature for the colorimetric reaction is from room temperature to 37°C.

7. The highly sensitive and rapid method for detecting total bacterial count integrating short-time culture and signal enhancement as described in claim 1, characterized in that, Step S4 establishes a semi-quantitative interpretation interval based on grayscale value analysis: For soy sauce samples, a grayscale value < 3100 corresponds to a total bacterial count < 100 CFU / mL, a grayscale value of 3100–4000 corresponds to a total bacterial count of 100–1000 CFU / mL, and a grayscale value > 4000 corresponds to a total bacterial count > 1000 CFU / mL; for wheat grain samples, a grayscale value < 1500 corresponds to a total bacterial count < 1000 CFU / mL, a grayscale value of 1500–3600 corresponds to a total bacterial count of 1000–10000 CFU / mL, and a grayscale value > 3600 corresponds to a total bacterial count > 10000 CFU / mL.

8. The highly sensitive and rapid detection method for total bacterial count integrating short-time culture and signal enhancement according to claim 1, characterized in that, The lateral flow test strip is assembled from a glass fiber membrane, a nitrocellulose membrane, and an absorbent pad.

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