Screening method for microbial sensitivity challenge test

Abstract

The invention discloses a screening method for a microbial sensitivity challenge test. The method comprises the following steps: respectively inoculating different types of microorganisms into a to-be-detected sample, transferring the to-be-detected sample into a sterile bottle with COsensing gel and a color indicator at the bottom, and culturing the to-be-detected sample; by monitoring the optical signal change of the indicator caused by COadsorption, the sensitivity of a sample to microorganisms is analyzed, and then the microorganism risk degree is systematically evaluated. According to the method, rapid, systematic and quantitative comprehensive evaluation on the risk degree of the microorganisms in the sample is realized, the application range is wide, the detection sensitivity is high, the time consumed for screening and judging is short, and an efficient and visual scientific basis is provided for the evaluation of the antimicrobial capacity of a product formula, the optimization of a sterilization process and the decision-making of quality control.

Description

Technical Field

[0001] This application relates to the field of microbial detection technology, specifically to a screening method for microbial sensitivity challenge testing. Background Technology

[0002] Currently, the common method for screening beverages for microbial risks involves inoculating samples with a quantitative amount of target bacterial solution and culturing them at a specific temperature. Samples are then taken at regular intervals for inoculation and culturing in traditional petri dishes. After the petri dishes have completed culturing, observation and counting are performed. The entire process is cumbersome, has a long testing cycle, and involves a large amount of engineering work.

[0003] While turbidimetric methods (OD values) are currently used to monitor biological growth, the turbidity (optical density, OD value) of the culture medium increases as microorganisms or cells grow and multiply in it. By illuminating the sample with light of a specific wavelength and measuring the intensity of transmitted or scattered light after passing through the culture medium, changes in biomass in the sample can be indirectly reflected. However, this method has very high requirements for the sample matrix (sample background); complex systems such as turbid juices, colored liquids, liquids containing particles, and emulsions cannot be used.

[0004] Some reports describe rapid detection of microorganisms in beverages using the adsorption and color change of carbon dioxide by sensing hydrogel materials, solving the problems of long detection time, complex operation, high cost, and insufficient sensitivity. This method is used for quality control on production lines and finished product inspection of various beverage products. Other reports use a combination of sensing hydrogel materials and a constant temperature incubator. The hydrogel's adsorption and color change in response to carbon dioxide rapidly indicates the presence and content of microorganisms. This method involves placing the beverage sample in the incubator, inserting the sensing hydrogel into the bottom of the culture bottle, and then attaching the sensing hydrogel to the top of the culture dish. After two days of incubation, the color change of the sensing hydrogel is observed to determine the presence and content of microorganisms. While these sensing hydrogel-based methods can achieve rapid detection of microorganisms in beverage samples, their core limitation is that they only confirm the presence of microorganisms in the product, rather than systematically determining the types of risky microorganisms. They cannot assess the risk status of the product itself after microbial contamination, thus failing to provide a comprehensive and efficient evaluation of the overall microbial risk level of the product. This makes it difficult for product developers to quickly and intuitively assess the overall antimicrobial capability of product formulations and to conduct precise sterilization condition optimization and risk assessment accordingly. Summary of the Invention

[0005] Therefore, it is necessary to provide a screening method for microbial sensitivity challenge testing.

[0006] The first aspect of this application provides a screening method for a microbial susceptibility challenge test, comprising the following steps:

[0007] Different types of microorganisms were inoculated into the sample to be tested and mixed thoroughly.

[0008] The mixed sample is transferred to a sterile bottle for incubation and testing to obtain the test results. The bottom of the sterile bottle is equipped with a sensor, which includes a sensing gel material that can adsorb CO2 and a color indicator. The color indicator displays a corresponding color change as the amount of CO2 adsorbed changes.

[0009] The test results are analyzed to determine whether the sample to be tested is sensitive to the microorganism.

[0010] Based on the sensitivity results of the test sample to different types of microorganisms, the degree of microbial risk of the test sample is determined.

[0011] In some embodiments, the microorganisms include one or more of bacteria, yeast, and mold;

[0012] Optionally, the bacteria include one or more of spore-forming and non-spore-forming bacteria; optionally, the spore-forming bacteria include one or more of Bacillus cereus, Bacillus subtilis, Bacillus coagulans, and Bacillus cyclophosphamide; the non-spore-forming bacteria include one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa.

[0013] The yeast includes one or more of Saccharomyces cerevisiae and Pichia pastoris;

[0014] The molds include one or more of Aspergillus niger, Penicillium, and Rhizopus.

[0015] In some embodiments, before inoculating the microorganisms into the sample to be tested and mixing them, the microorganisms are cultured in a culture medium to the logarithmic growth phase and then prepared into a bacterial suspension.

[0016] In some embodiments, the concentration of the bacterial suspension is 10. 0 CFU / mL ~10 8 CFU / mL.

[0017] In some embodiments, the concentration of the microbial inoculum is 10. 0 CFU / mL ~10 8 CFU / mL.

[0018] In some implementations, the sensor satisfies one or more of the following conditions:

[0019] The sensor consists of a sensing gel material capable of adsorbing CO2 and a color indicator;

[0020] The weight ratio of the CO2-adsorbing sensing gel material to the color indicator is (100~500):1;

[0021] The thickness of the sensor is 3mm to 8mm.

[0022] In some embodiments, the culture and detection step includes transferring the mixed sample to a sterile bottle; placing the sterile bottle in an automated microbial culture and detection system for culture and detection, the automated microbial culture and detection system being equipped with an emission light source and a photon detector to monitor changes in the optical signal of the sensor in real time.

[0023] In some embodiments, the volume of the mixed sample to be transferred is 10 mL to 100 mL.

[0024] In some implementations, the cultivation step includes shaking cultivation at a constant temperature.

[0025] In some implementations, the cultivation conditions include: a shaking speed of 80 rpm to 400 rpm and a cultivation temperature of 20°C to 45°C.

[0026] In some embodiments, the method further includes transferring the uninoculated test sample to the sterile bottle for culture and testing, serving as a negative control sample.

[0027] In some implementations, the criteria for analyzing the detection results and determining whether the sample to be tested is sensitive to the microorganism are as follows:

[0028] When the slope of the change in reflectance value is greater than or equal to 0.3 RU / min for 3 to 5 consecutive unit time periods, the test sample is sensitive to the microorganism; or, if the acceleration of the change in reflectance value of the test sample inoculated with the microorganism is greater than or equal to 0.02 RU / min², and the acceleration of the change in reflectance value of the negative control sample is less than 0.02 RU / min², the test sample is sensitive to the microorganism.

[0029] In some implementations, the criteria for determining the microbial risk level of a test sample based on its sensitivity to different types of microorganisms are as follows:

[0030] When the sample to be tested is sensitive to all of the inoculated spore-forming bacteria, non-spore-forming bacteria, molds, and yeasts, the sample has a high level of microbial risk.

[0031] When the sample to be tested is sensitive to all of the inoculated spore-forming bacteria, molds and yeasts, and is not sensitive to the inoculated non-spore-forming bacteria, the microbial risk level of the sample to be tested is moderate to high.

[0032] When the sample to be tested is not sensitive to either spore-forming or non-spore-forming bacteria, but is sensitive to inoculated molds and yeasts, the microbial risk level of the sample to be tested is moderate.

[0033] When the sample to be tested is not sensitive to the inoculated spore-forming bacteria, non-spore-forming bacteria, molds, and yeasts, the microbial risk level of the sample to be tested is low.

[0034] In some embodiments, the sample to be tested includes one or more of beverages, food, cosmetics, and pharmaceuticals;

[0035] Optionally, the sample to be tested is a beverage, which includes one or more of acidic beverages, neutral beverages, bottled water, natural water, and mineral water.

[0036] The aforementioned method enables a rapid, systematic, and quantitative comprehensive assessment of the microbial risk level of samples. It has a wide range of applications, high detection sensitivity, and short screening and judgment time, providing efficient and intuitive scientific basis for product formulation antimicrobial capability assessment, sterilization process optimization, and quality control decisions. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0038] Figure 1 This is a schematic diagram of the microbial sensitivity challenge test screening process in one embodiment of this application;

[0039] Figure 2 This is a graph showing the trend of RU values ​​of different strains over time in Example 1 of this application;

[0040] Figure 3 This is a graph showing the trend of RU values ​​of different strains over time in Example 2 of this application. Detailed Implementation

[0041] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0044] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0045] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0046] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0047] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0048] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0049] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0050] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0051] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0052] Currently, methods based on sensing hydrogels can achieve rapid detection of microorganisms in samples such as beverages. However, their core limitation lies in the fact that they only confirm whether a product contains microorganisms, rather than systematically determining the types of risky microorganisms. They cannot assess the risk status of a product after contamination with microorganisms, thus hindering a comprehensive and efficient evaluation of the overall microbial risk level of the product. This makes it difficult for product developers to quickly and intuitively evaluate the overall antimicrobial capability of their product formulations and, based on this, to accurately optimize sterilization conditions and conduct risk assessments.

[0053] Based on this, the embodiments of this application at least provide a screening method for microbial sensitivity challenge testing.

[0054] In some implementations, a product microbial sensitivity challenge test method is provided that is widely applicable, highly sensitive, and quick to screen and determine, in order to meet the needs of determining the risk of newly developed food, beverage, cosmetic and other samples being infected with microorganisms during the production process and to formulate product sterilization conditions.

[0055] In some implementations, carbon dioxide released by microbial growth and metabolism diffuses and permeates into the sensing gel material at the bottom of the bottle, forming carbonic acid. This causes a local pH decrease, which in turn triggers a reversible color change in the color indicator. The higher the microbial content or the weaker the sample's ability to inhibit it (i.e., the more sensitive it is), the faster the microbial growth, and the faster and earlier the rate of color change (such as the slope of reflectance change) caused by its metabolic acid production. By monitoring the dynamic changes of these optical signals, a rapid and quantitative assessment of the sample's sensitivity can be achieved.

[0056] In a first aspect of this application, a screening method for a microbial sensitivity challenge test is provided, in which different types of microorganisms are inoculated into the sample to be tested and transferred to a sterile bottle with a CO2 sensing gel and a color indicator at the bottom for incubation; by monitoring the changes in the optical signal of the indicator caused by CO2 adsorption, the sensitivity of the sample to microorganisms is analyzed, and then the degree of microbial risk is systematically assessed.

[0057] In some implementations, the following steps are included:

[0058] S100: Inoculate different types of microorganisms into the sample to be tested and mix them thoroughly;

[0059] S200: The mixed sample to be tested is transferred to a sterile bottle for incubation and testing to obtain the test results; a sensor is installed at the bottom of the sterile bottle, which includes a sensing gel material that can adsorb CO2 and a color indicator. The color indicator displays the corresponding color change as the amount of CO2 adsorbed changes.

[0060] S300: Analyze the test results to determine whether the sample is sensitive to microorganisms;

[0061] S400: Determine the microbial risk level of the sample based on its sensitivity to different types of microorganisms.

[0062] In some implementations, the sample to be tested includes, but is not limited to, one or more of beverages, food, cosmetics, and pharmaceuticals.

[0063] In some implementations, the sample to be tested is a beverage, and further, the beverage includes, but is not limited to, one or more of acidic beverages, neutral beverages, bottled water, natural water, and mineral water.

[0064] In some implementations, the microorganisms include one or more of bacteria, yeast, and mold.

[0065] In some embodiments, the bacteria include one or more of spore-forming and non-spore-forming bacteria. Further, the spore-forming bacteria include, but are not limited to, one or more of Bacillus cereus, Bacillus subtilis, Bacillus coagulans, and Bacillus cyclophosphamide; the non-spore-forming bacteria include, but are not limited to, one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa.

[0066] In some implementations, the yeast includes, but is not limited to, one or more of Saccharomyces cerevisiae and Pichia pastoris.

[0067] In some embodiments, the mold includes, but is not limited to, one or more of Aspergillus niger, Penicillium, and Rhizopus.

[0068] In some embodiments, before step S100, the microorganisms are cultured in a culture medium to the logarithmic growth phase and then prepared into a bacterial suspension.

[0069] In some embodiments, the concentration of the bacterial suspension is 10. 0 CFU / mL ~10 8 CFU / mL. Without limitation, the concentration of the bacterial suspension can be, but is not limited to, 10⁻⁶ CFU / mL. 0 CFU / mL, 10 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL or a value or range between any two of the above values. Further, when the microorganism is bacteria, the concentration of the bacterial suspension is 10. 7 CFU / mL ~10 8 CFU / mL; when the microorganism is a fungus, the concentration of the bacterial suspension is 10. 4 CFU / mL ~10 6 CFU / mL.

[0070] In some embodiments, in step S100, the concentration of the microbial inoculum is 10. 0 CFU / mL ~10 8 CFU / mL. Without limitation, the concentration of the microbial inoculum can be, but is not limited to, 10⁻⁶ CFU / mL. 0 CFU / mL, 10 1 CFU / mL, 10 2 CFU / mL, 10 3CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL or any value or range between the two values ​​mentioned above.

[0071] In some embodiments, in step S200, the sensor consists of a sensing gel material capable of adsorbing CO2 and a color indicator. It should be noted that this sensor can be prepared using conventional methods in the art, such as mixing the sensing gel material and the color indicator in a certain proportion and then solidifying them at the bottom of a sterile bottle.

[0072] In some embodiments, the sensing gel material capable of adsorbing CO2 includes, but is not limited to, natural polymer gels, synthetic polymer gels, etc.

[0073] In some embodiments, the color indicator includes one or more of pH-sensitive dyes, metal ion complexes, and fluorescent probes. Further, the pH-sensitive dyes include, but are not limited to, bromothymol blue, bromophenol blue, and methyl red; the metal ion complexes include, but are not limited to, cobalt chloride hexahydrate and cobalt-ethylenediamine complexes; and the fluorescent probes include, but are not limited to, fluorescein isothiocyanate and spiropyran derivatives.

[0074] In some embodiments, the weight ratio of the CO2-adsorbing gel system to the color indicator is (10~500):1. Non-limitingly, the weight ratio of the CO2-adsorbing gel system to the color indicator can be, but is not limited to, 10:1, 50:1, 100:1, 200:1, 300:1, 400:1, 500:1, or any ratio or range between two of the above.

[0075] It should be noted that the CO2-adsorbing sensing gel materials and color indicators include, but are not limited to, the combinations shown in Table 1 below:

[0076] Table 1

[0077]

[0078] In some embodiments, the thickness of the sensor is 3mm to 8mm. Non-limitingly, the thickness of the sensor can be, but is not limited to, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, or any value or range between two of the above.

[0079] In some embodiments, in step S200, the culture and detection step includes transferring the mixed sample to a sterile bottle; placing the sterile bottle in an automated microbial culture and detection system for culture and detection. The automated microbial culture and detection system is equipped with an emission light source and a photon detector, and monitors the changes in the optical signal of the sensor in real time by detecting the intensity of reflected light on the surface of the sensor.

[0080] It should be noted that the automated microbial culture and detection system indirectly reflects the growth status of microorganisms by monitoring changes in the intensity of reflected light on the sensor surface. The principle is as follows: carbon dioxide produced by microbial metabolism is adsorbed by the sensing gel, causing a change in the pH value within the gel, which in turn triggers a reversible or irreversible color change in the color indicator. This color change leads to a change in the intensity of light reflected from the sensor surface at a specific wavelength. By measuring this change in reflectance intensity in real time (i.e., reflectance value, RU) and analyzing its rate of change over time (such as slope or acceleration), the system can dynamically determine whether microorganisms are growing and multiplying in the sample, thereby assessing the sample's sensitivity to microorganisms.

[0081] In some embodiments, the volume of the mixed sample transferred is 10 mL to 100 mL. Non-limitingly, the volume of the mixed sample transferred can be, but is not limited to, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, or any value or range between two of the above.

[0082] In some embodiments, the cultivation step includes shaking cultivation at a constant temperature; further, the cultivation conditions include: a shaking speed of 80 rpm to 400 rpm and a cultivation temperature of 20°C to 45°C. Non-limitingly, the shaking speed can be, but is not limited to, 80 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, or any value or range between two of the above values; the cultivation temperature can be, but is not limited to, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or any value or range between two of the above values.

[0083] In some implementations, when the microorganism is bacteria, the culture temperature is 30-45°C.

[0084] In some implementations, when the microorganism is a fungus, the culture temperature is 20-30°C.

[0085] Understandably, the incubation temperature and duration can be adjusted based on the specific target microorganisms being tested, as well as the actual conditions of the samples being evaluated during production and storage.

[0086] In some embodiments, step S200 further includes transferring the uninoculated test sample to a sterile bottle for culture and testing, serving as a negative control sample. It is understood that the negative control sample and the test sample are cultured under the same conditions.

[0087] In some embodiments, in step S300, the criteria for determining whether the test sample is sensitive to microorganisms are: when the slope of the change in reflectance value is greater than or equal to 0.3RU / min for 3 to 5 consecutive unit time periods, the test sample is sensitive to microorganisms; and / or, if the acceleration of the change in reflectance value of the test sample inoculated with microorganisms is greater than or equal to 0.02RU / min², and the acceleration of the change in reflectance value of the negative control sample is less than 0.02RU / min², the test sample is sensitive to microorganisms.

[0088] In some implementations, in step S400, the criteria for determining the microbial risk level of the sample to be tested, based on the sensitivity results of the sample to different types of microorganisms, are as follows:

[0089] When the sample to be tested is sensitive to all of the inoculated spore-forming bacteria, non-spore-forming bacteria, molds, and yeasts, the sample has a high level of microbial risk.

[0090] When the sample to be tested is sensitive to all of the inoculated spore-forming bacteria, molds and yeasts, but not sensitive to the inoculated non-spore-forming bacteria, the microbial risk level of the sample to be tested is moderate to high.

[0091] When the sample to be tested is not sensitive to either spore-forming or non-spore-forming bacteria, but is sensitive to inoculated molds and yeasts, the microbial risk level of the sample to be tested is moderate.

[0092] When the sample to be tested is not sensitive to the inoculated spore-forming bacteria, non-spore-forming bacteria, molds, and yeasts, the microbial risk level of the sample to be tested is low.

[0093] The following are some examples.

[0094] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0095] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0096] The following embodiments provide a method for microbial sensitivity challenge testing, and the flowchart is shown below. Figure 1 As shown, the specific steps include:

[0097] 1. Selection of Challenge Strains. Select representative microbial strains relevant to the product being tested, typically including bacteria, yeasts, and molds. For example:

[0098] Bacteria: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, Bacillus coagulans, Bacillus cyclophosphamide, etc.

[0099] Yeast: Saccharomyces cerevisiae, Pichia pastoris, etc.

[0100] Molds: Aspergillus niger, Penicillium, Rhizopus, etc.

[0101] 2. Inoculation and Culture. After obtaining the pure target strain using the streak plating method, enrichment culture was performed. The bacterial concentration was prepared to 10⁻⁶. 8 CFU / mL, fungal concentration 10 5 After preparing the bacterial suspension at CFU / mL, add the bacterial suspension to the sample to be evaluated and mix thoroughly.

[0102] Transfer 10 mL to 100 mL of sample to a sterile bottle containing a sensor consisting of a gel system that can adsorb CO2 and a color indicator at the bottom. Incubate at a constant temperature and a constant speed of shaking at 80 to 400 rpm.

[0103] An automated microbial culture and detection system equipped with an emission light source and photon detector monitors the sensor color, and the rate of color change determines whether the target microorganism will multiply in the sample. If the target microorganism is bacteria, the culture temperature is 30–45°C; if the target microorganism is fungi, the culture temperature is 20–30°C. The culture temperature and duration are determined by the target microorganism and the production and storage conditions of the sample to be evaluated. Negative control samples are cultured together with the test samples in the same manner.

[0104] 3. Sample microbial sensitivity identification.

[0105] The sensitivity of a product to a target microorganism is determined by calculating the slope of the change in reflectance value per unit time (ΔRU / Δt) and the second derivative of the reflectance value change (acceleration). When the slope exceeds the dynamic rate threshold (>0.3 RU / min) for 3-5 consecutive unit time periods, it indicates that the microorganism has entered the logarithmic growth phase, and the sample is considered sensitive to the inoculated microorganism. Compared with negative samples, if the acceleration of a positive sample consistently exceeds the acceleration threshold of 0.02 RU / min², and the acceleration of a negative sample consistently falls below the acceleration threshold of 0.02 RU / min², the sample is considered sensitive to the inoculated microorganism; if the acceleration of both positive and negative samples consistently exceeds the acceleration threshold (0.02 RU / min²), the sample is considered insensitive to the inoculated microorganism.

[0106] Based on a comprehensive assessment of the sensitivity of various microorganisms in the product: if spore-forming bacteria, non-spore-forming bacteria, molds, and yeasts do not proliferate in the product, the product has a low microbial risk; if spore-forming bacteria and non-spore-forming bacteria do not proliferate in the product, but molds and yeasts do, the product has a moderate microbial risk; if spore-forming bacteria, molds, and yeasts proliferate in the product, but non-spore-forming bacteria do not, the product has a moderately high microbial risk; if spore-forming bacteria, non-spore-forming bacteria, molds, and yeasts all proliferate in the product, the product has an extremely high microbial risk.

[0107] Example 1

[0108] Microbial sensitivity test screening of a beverage

[0109] Fresh colonies of Klebsiella pneumoniae, Bacillus cereus, Saccharomyces cerevisiae, and Aspergillus niger were cultured separately. A portion of the bacteria was taken using a sterile inoculation loop and added to 10 mL of sterile water, vortexed to mix, and a bacterial concentration of 10⁻⁶ was prepared. 8 A bacterial suspension with CFU / mL and a yeast concentration of 10... 5 CFU / mL bacterial suspension. Take 100 μL of bacterial suspension and inoculate it into the sample and mix well. Take 10 mL of sample and transfer it to a sterile bottle containing a sensor consisting of a gel system with CO2 adsorption capacity at the bottom and a color indicator. Place the bottle in an automated microbial culture and detection system equipped with an emission light source and a photon detector to monitor the sensor color. Incubate at 30°C with uniform shaking at 150 rpm.

[0110] Negative control test: Samples without the added strain were added to a sterile bottle equipped with a sensor and placed in the detection system under the same conditions. RU values ​​were continuously recorded at 10-minute intervals. Positive strain test: Samples with the added strain were added to a sterile bottle equipped with a sensor and placed in the detection system under the same conditions. RU values ​​were continuously recorded. The start and end times of the logarithmic growth phase for each positive strain were identified using data software, and the slope of RU change during this phase was calculated. When the slope exceeds the dynamic rate threshold (>0.3 RU / min) for three consecutive measurement units, or when the acceleration of the positive sample consistently exceeds the acceleration threshold of 0.02 RU / min², and the acceleration of the negative sample consistently falls below the acceleration threshold of 0.02 RU / min², it indicates that the microorganism has entered the logarithmic growth phase, and the sample is deemed sensitive to the inoculated microorganism. Detection results are as follows: Figure 2 As shown, the slope of Aspergillus niger, Bacillus cereus, and Saccharomyces cerevisiae for three consecutive test units > 0.3 RU / min indicates that these three microorganisms have entered the logarithmic growth phase and the sample is sensitive to these three microorganisms. The slope of Klebsiella pneumoniae for three consecutive test units < 0.3 RU / min indicates that the sample is not sensitive to this microorganism. Therefore, the microbial risk of this product is judged to be moderate to high.

[0111] Example 2

[0112] Microbial sensitivity test screening of a beverage

[0113] Fresh colonies of Klebsiella pneumoniae, Bacillus cereus, Saccharomyces cerevisiae, and Aspergillus niger were cultured separately. A portion of the bacteria was taken using a sterile inoculation loop and added to 10 mL of sterile water, vortexed to mix, and a bacterial concentration of 10⁻⁶ was prepared. 8 A bacterial suspension with CFU / mL and a yeast concentration of 10... 5 CFU / mL bacterial suspension. Take 100 μL of bacterial suspension and inoculate it into the sample and mix well. Take 10 mL of sample and transfer it to a sterile bottle containing a sensor consisting of a gel system with CO2 adsorption capacity at the bottom and a color indicator. Place the bottle in an automated microbial culture and detection system equipped with an emission light source and a photon detector to monitor the sensor color. Incubate at 30°C with uniform shaking at 150 rpm.

[0114] Negative control test: Samples without the added strain were added to a sterile bottle equipped with a sensor and placed in the detection system under the same conditions. RU values ​​were continuously recorded at 10-minute intervals. Positive strain test: Samples with the added strain were added to a sterile bottle equipped with a sensor and placed in the detection system under the same conditions. RU values ​​were continuously recorded. The start and end times of the logarithmic growth phase for each positive strain were identified using data software, and the slope of RU change during this phase was calculated. When the slope exceeds the dynamic rate threshold (>0.3 RU / min) for three consecutive measurement units, or when the acceleration of the positive sample consistently exceeds the acceleration threshold of 0.02 RU / min², and the acceleration of the negative sample consistently falls below the acceleration threshold of 0.02 RU / min², it indicates that the microorganism has entered the logarithmic growth phase, and the sample is deemed sensitive to the inoculated microorganism. Detection results are as follows: Figure 3 As shown, the slope of Aspergillus niger and Saccharomyces cerevisiae for three consecutive test units is >0.3RU / min, indicating that these two microorganisms have entered the logarithmic growth phase and the sample is sensitive to these two microorganisms. The slope of Bacillus cereus and Klebsiella pneumoniae for three consecutive test units is <0.3RU / min, indicating that the sample is not sensitive to these two microorganisms. Therefore, the product is judged to have a moderate microbial risk.

[0115] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0116] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A screening method for microbial susceptibility challenge testing, characterized in that, Includes the following steps: Different types of microorganisms were inoculated into the sample to be tested and mixed thoroughly. The mixed sample is transferred to a sterile bottle for incubation and testing to obtain the test results. The bottom of the sterile bottle is equipped with a sensor, which includes a sensing gel material that can adsorb CO2 and a color indicator. The color indicator displays a corresponding color change as the amount of CO2 adsorbed changes. The test results are analyzed to determine whether the sample to be tested is sensitive to the microorganism. Based on the sensitivity results of the test sample to different types of microorganisms, the degree of microbial risk of the test sample is determined.

2. The screening method for microbial susceptibility challenge testing as described in claim 1, characterized in that, The microorganisms include one or more of bacteria, yeast, and mold; Optionally, the bacteria include one or more of spore-forming and non-spore-forming bacteria; the spore-forming bacteria include one or more of Bacillus cereus, Bacillus subtilis, Bacillus coagulans, and Bacillus cyclophosphamide; the non-spore-forming bacteria include one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. The yeast includes one or more of Saccharomyces cerevisiae and Pichia pastoris; The molds include one or more of Aspergillus niger, Penicillium, and Rhizopus.

3. The screening method for microbial susceptibility challenge testing as described in claim 1, characterized in that, Before inoculating the microorganisms into the sample to be tested and mixing them, the microorganisms are also cultured in a culture medium to the logarithmic growth phase and then prepared into a bacterial suspension. Optionally, the concentration of the bacterial suspension is 10. 0 CFU / mL ~10 8 CFU / mL.

4. The screening method for microbial susceptibility challenge testing as described in claim 3, characterized in that, The concentration of the microbial inoculation is 10. 0 CFU / mL ~10 8 CFU / mL.

5. The screening method for microbial susceptibility challenge testing as described in claim 1, characterized in that, The sensor satisfies one or more of the following conditions: The sensor consists of a sensing gel material capable of adsorbing CO2 and a color indicator; The weight ratio of the CO2-adsorbing sensing gel material to the color indicator is (100~500):1; The thickness of the sensor is 3mm to 8mm.

6. The screening method for microbial susceptibility challenge testing as described in claim 1, characterized in that, The culture and detection steps include: transferring the mixed sample to a sterile bottle; placing the sterile bottle in an automated microbial culture and detection system for culture and detection, wherein the automated microbial culture and detection system is equipped with an emission light source and a photon detector to monitor the changes in the optical signal of the sensor in real time; Optionally, the volume of the mixed sample to be transferred is 10 mL to 100 mL; Optionally, the cultivation step includes shaking cultivation at a constant temperature; Further optionally, the culture conditions include: a shaking speed of 80 rpm to 400 rpm and a culture temperature of 20°C to 45°C.

7. The screening method for microbial susceptibility challenge testing as described in claim 6, characterized in that, It also includes transferring uninoculated microbial test samples to the sterile bottle for culture and testing, serving as negative control samples.

8. The screening method for microbial susceptibility challenge testing as described in claim 7, characterized in that, The criteria for determining whether the test sample is sensitive to the microorganism by analyzing the test results are as follows: When the slope of the change in reflectance value is greater than or equal to 0.3 RU / min for 3 to 5 consecutive unit time periods, the test sample is sensitive to the microorganism; or, if the acceleration of the change in reflectance value of the test sample inoculated with the microorganism is greater than or equal to 0.02 RU / min², and the acceleration of the change in reflectance value of the negative control sample is less than 0.02 RU / min², the test sample is sensitive to the microorganism.

9. The screening method for microbial susceptibility challenge testing as described in claim 8, characterized in that, Based on the sensitivity results of the test sample to different types of microorganisms, the criteria for judging the microbial risk level of the test sample are as follows: When the sample to be tested is sensitive to all of the inoculated spore-forming bacteria, non-spore-forming bacteria, molds, and yeasts, the sample has a high level of microbial risk. When the sample to be tested is sensitive to all of the inoculated spore-forming bacteria, molds and yeasts, and is not sensitive to the inoculated non-spore-forming bacteria, the microbial risk level of the sample to be tested is moderate to high. When the sample to be tested is not sensitive to both non-spore-forming and spore-forming bacteria inoculated with the inoculated molds and yeasts, the microbial risk level of the sample to be tested is moderate. When the sample to be tested is not sensitive to both non-spore-forming and spore-forming bacteria, molds, and yeasts inoculated, the microbial risk level of the sample to be tested is low.

10. The screening method for microbial susceptibility challenge testing as described in any one of claims 1 to 9, characterized in that, The samples to be tested include one or more of beverages, food, cosmetics, and pharmaceuticals; Optionally, the sample to be tested is a beverage, which includes one or more of acidic beverages, neutral beverages, bottled water, natural water, and mineral water.

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