Method for testing microorganism invasion of packaging material

Abstract

The invention discloses a method for detecting microorganism invasion of a packaging material, and belongs to the technical field of packaging material detection. The detection method provided by the invention can simulate the sealing property and the microbial invasion risk of the packaging material in an actual transportation scene, so that the pollution detection has higher specificity and visualization effects. Compared with a traditional detection method, the detection method has the advantages that the detection sensitivity and accuracy are improved, interference of background bacteria can be effectively eliminated, the performance of the packaging material in the aspect of microbial protection is truly reflected, the pollution path recognition capability is improved, and the detection method is suitable for popularization and application. A scientific and reliable technical means is provided for verification of package integrity of beverages, bottled water and the like, and the shelf life and safety of products can be improved.

Description

Technical Field

[0001] This invention relates to the field of packaging material testing technology, and in particular to a method for testing microbial intrusion into packaging materials. Background Technology

[0002] Currently, the main microbiological testing methods for the airtightness of packaging materials are as follows: (1) Bubble test: The packaging material is immersed in water, and the airtightness is judged by observing whether bubbles are generated. This method is simple to operate, but has low sensitivity and is difficult to detect minute leaks. (2) Dye penetration test: The packaging material is immersed in a specific dye solution, and the airtightness is judged by observing whether the dye penetrates into the packaging. This method has high sensitivity, but may cause contamination to the packaging material. (3) Pressure difference test: The airtightness is judged by applying different pressures inside and outside the packaging and observing the pressure changes. This method has high sensitivity, but the equipment is more complex.

[0003] Currently, water-based beverage products widely use plastic bottles with plastic screw caps or heat-sealed caps. However, in actual production and distribution processes, factors such as tiny gaps between the cap and the bottle opening, unstable sealing strength, or vibrations during transportation may allow microorganisms from the environment to enter through these gaps, causing product contamination and affecting shelf life and safety.

[0004] Existing commonly used methods for evaluating packaging airtightness, such as negative pressure testing and water bath leakage testing, mainly assess physical sealing performance and are difficult to accurately reflect the packaging's protective effect against microbial intrusion. Furthermore, some testing methods use vegetative bacteria or contaminating bacteria that exhibit rapid growth and significant experimental interference, making them unsuitable as stable indicators for simulating microbial intrusion.

[0005] Therefore, there is an urgent need for a packaging seal testing method based on microbial bioindicators that can simulate the risk of contamination in real storage and transportation scenarios, especially one that can effectively evaluate the protective effect of bottle cap structure against contaminants such as Pseudomonas aeruginosa. Summary of the Invention

[0006] The main objective of this invention is to provide a method for testing microbial intrusion in packaging materials, thereby solving the technical problem that existing methods for evaluating the sealing performance of packaging materials are insufficient to reflect the protective effect of packaging materials against microbial intrusion.

[0007] To achieve the above objectives, the present invention provides a method for testing microbial intrusion in packaging materials, comprising the following steps:

[0008] S10. Identify the contamination indicator bacteria and prepare an indicator bacteria spore suspension from the contamination indicator bacteria;

[0009] S20. Fill the packaging material to be tested with sterile liquid;

[0010] S30. Inject the indicator bacteria spore suspension into the sealed part of the packaging material to be tested, and then seal the sealed part.

[0011] S40. Conduct transportation simulation on the sealed packaging materials to be tested;

[0012] S50. Extract the liquid from the inside of the test packaging material after the transportation simulation is completed, filter and culture the extracted liquid, and detect the presence of the contamination indicator bacteria to evaluate the airtightness of the test packaging material.

[0013] In some embodiments of the present invention, the contamination indicator bacteria include Bacillus thermophilus.

[0014] In some embodiments of the present invention, the colony-forming units of the indicator bacterial spore suspension are 10-1. 7 CFU / mL.

[0015] In some embodiments of the present invention, the spores of the indicator bacterial spore suspension are labeled with a fluorescent dye.

[0016] In some embodiments of the present invention, the fluorescent dye includes fluorescein isothiocyanate or rhodamine B.

[0017] In some embodiments of the present invention, the sealing part is the junction between the bottle cap and the edge of the bottle mouth.

[0018] In some embodiments of the present invention, the transportation simulation includes: placing the sealed packaging material to be tested on a transport vehicle and transporting it for 70 to 75 hours at a transport temperature of 20°C to 30°C.

[0019] In some embodiments of the present invention, the step of extracting the liquid includes: piercing the sealed portion of the packaging material to be tested with a sterile sampling needle and extracting the liquid inside the packaging material to be tested.

[0020] In some embodiments of the present invention, the detection of the presence of contamination indicator bacteria includes the following steps: passing the extracted liquid through a 0.22 μm filter membrane, placing the filter membrane in a culture medium for incubation, and observing whether the contamination indicator bacteria appear.

[0021] In some embodiments of the present invention, the extracted liquid is passed through a 0.22 μm filter membrane, and the appearance of a fluorescent signal on the filter membrane is observed. Then, the filter membrane is placed in a culture medium for incubation, and the presence of the contamination indicator bacteria is observed.

[0022] The beneficial effects that this invention can achieve are:

[0023] The testing method of this invention can simulate the sealing performance of packaging materials and the risk of microbial intrusion, such as the risk of contamination by Pseudomonas aeruginosa, under actual transportation scenarios, and makes contamination detection more specific and visual. Compared with traditional detection methods, this method not only improves the sensitivity and accuracy of detection, but also effectively eliminates the interference of background bacteria, truly reflects the performance of packaging materials in terms of microbial protection, enhances the ability to identify contamination pathways, and provides a scientific and reliable technical means for verifying the integrity of packaging such as beverages and bottled water, thus helping to improve product shelf life and safety. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0025] Figure 1 This is a flowchart of a method for testing microbial invasion of packaging materials according to an embodiment of the present invention;

[0026] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0027] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] In this invention, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature. Furthermore, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. If the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0030] Existing commonly used methods for evaluating packaging airtightness, such as negative pressure testing and water bath leakage testing, mainly assess physical sealing performance and are difficult to accurately reflect the packaging's protective effect against microbial intrusion. Furthermore, some testing methods use vegetative bacteria or contaminating bacteria that exhibit rapid growth and significant experimental interference, making them unsuitable as stable indicators for simulating microbial intrusion.

[0031] Therefore, there is an urgent need for a packaging seal testing method based on microbial bioindicators that can simulate the risk of contamination in real storage and transportation scenarios, especially one that can effectively evaluate the protective effect of bottle cap structure against contaminants such as Pseudomonas aeruginosa.

[0032] In view of this, such as Figure 1 As shown, the present invention provides a method for testing microbial intrusion in packaging materials, comprising the following steps:

[0033] S10. Identify the contamination indicator bacteria and prepare an indicator bacteria spore suspension from the contamination indicator bacteria;

[0034] S20. Fill the packaging material to be tested with sterile liquid;

[0035] S30. Inject the indicator bacteria spore suspension into the sealed part of the packaging material to be tested, and then seal the sealed part.

[0036] S40. Conduct transportation simulation on the sealed packaging materials to be tested;

[0037] S50. Extract the liquid from the inside of the test packaging material after the transportation simulation is completed, filter and culture the extracted sterile liquid, and detect the presence of contamination indicator bacteria to assess the airtightness of the test packaging material.

[0038] The testing method of this invention can simulate the sealing performance of packaging materials and the risk of microbial intrusion, such as the risk of contamination by Pseudomonas aeruginosa, under actual transportation scenarios, and makes contamination detection more specific and visual. Compared with traditional detection methods, this method not only improves the sensitivity and accuracy of detection, but also effectively eliminates the interference of background bacteria, truly reflects the performance of packaging materials in terms of microbial protection, enhances the ability to identify contamination pathways, and provides a scientific and reliable technical means for verifying the integrity of packaging such as beverages and bottled water, thus helping to improve product shelf life and safety.

[0039] In some embodiments, the contamination indicator bacteria include Bacillus thermophilus, whose optimal growth temperature is approximately 55°C, and which can effectively distinguish contamination from other bacteria in a normal temperature environment.

[0040] In some embodiments, an indicator spore suspension is obtained by inducing spore formation of contaminating indicator bacteria through heat shock and restricted nutrition.

[0041] In some embodiments, the colony-forming units of the indicator bacterial spore suspension are 10-1. 7 CFU / mL.

[0042] In some embodiments, the indicator bacterium is *Bacillus thermophilus*. *Bacillus thermophilus* is cultured at 55°C to the late logarithmic phase, then subjected to heat shock at 80°C and induced to form spores under restricted nutrient conditions. The spore pellet is then collected by centrifugation and resuspended in PBS buffer to 10⁻⁶. 7 A spore suspension was obtained at CFU / mL.

[0043] In some embodiments, the spores of contaminating indicator bacteria are labeled with fluorescent dyes. Staining the outer wall of spores in an indicator bacterial spore suspension with fluorescent dyes enables the visual tracking of contamination pathways.

[0044] In some embodiments, the fluorescent dye includes fluorescein isothiocyanate (FITC) or rhodamine B.

[0045] In some embodiments, the indicator bacterium spore suspension and the fluorescent dye solution are reacted at room temperature and in the dark for 25 to 35 minutes, with the room temperature being 20°C to 30°C. The reaction time can also be 30 minutes. After the reaction is completed, the mixture is washed to remove the free dye, thereby obtaining a fluorescently labeled indicator bacterium spore suspension. This fluorescently labeled indicator bacterium spore suspension is then injected into the sealed portion of the packaging material to be tested to simulate biological invasion.

[0046] In some embodiments, the sterile liquid includes sterile purified water.

[0047] In some embodiments, after filling the packaging material to be tested with liquid, the packaging material to be tested is placed in a sterile environment and left to stand for 48 hours to eliminate potential interfering bacteria in the environment.

[0048] In some embodiments, the packaging to be tested includes a bottle neck and a bottle cap, with the sealing area being the junction between the bottle cap and the edge of the bottle neck. After screwing the bottle cap onto the bottle neck, the bottle cap is punctured, and an indicator bacterial spore suspension is injected into the junction between the bottle cap and the edge of the bottle neck. The bottle cap is then immediately heat-sealed. Injecting the indicator bacterial spore suspension into this sealing area simulates microbial invasion, allowing for the testing of the bottle neck sealing performance and resistance to microbial invasion of the packaging material.

[0049] In some embodiments, the injection volume of the indicator bacterial spore suspension is 0.5 ml to 1.5 ml, and may be 1 ml.

[0050] Step S40 of the present invention simulates transportation of the packaging material to be tested, which can verify the ability of the packaging material's sealing performance to resist factors such as transportation vibration.

[0051] In some embodiments, the transportation simulation includes the following steps: placing the packaging material to be tested on a transport vehicle and transporting it for 70 to 75 hours, which may be 72 hours.

[0052] In some embodiments, the transportation environment is 20℃~30℃, simulating the impact of normal temperature transportation environment on the sealing performance of packaging materials, which is closer to real life and the test results are more valuable for reference.

[0053] In some embodiments, the step of extracting liquid includes: piercing the sealed portion of the packaging material to be tested with a sterile sampling needle to extract liquid from inside the packaging material to be tested.

[0054] In some embodiments, after the transportation simulation ends and before liquid extraction, the sealing area is disinfected. If the sealing area is the junction of the bottle cap and the bottle mouth edge, the outside of the bottle cap is disinfected. This reduces contamination from external microorganisms on the packaging material being tested, which could affect the test results.

[0055] The amount of liquid extracted depends on the specific circumstances; in some embodiments, the extraction volume is 200 ml.

[0056] In some embodiments, detecting the presence of contamination indicator bacteria includes the following steps: filtering the extracted liquid using a filter membrane, culturing the filtered filter membrane in a culture medium, and observing whether spores of indicator bacteria appear. If they appear, it indicates that the spores have entered the interior of the packaging material, indicating a risk of contamination at the sealed area and that the sealing performance of the packaging material is unqualified. If they do not appear, it indicates that the sealing performance of the sealed area of ​​the packaging material is qualified.

[0057] In some embodiments, detecting the presence of contamination indicator bacteria includes the following steps: the filter membrane has a pore size of 0.22 μm.

[0058] In some embodiments, the filter membrane is an aqueous filter membrane.

[0059] In some embodiments, the filter membrane is placed on a TSA plate at 55°C and cultured for 40 to 50 hours, or up to 48 hours.

[0060] In some embodiments, when fluorescent dyes are used to stain spores—that is, a fluorescently labeled spore suspension is injected into the sealed area to simulate biological invasion—in the subsequent steps of filtering and culturing the extracted liquid to detect the presence of contaminating indicator bacteria, the filter membrane is placed on a glass slide and irradiated with blue light under light-protected conditions. The appearance of fluorescence on the filter membrane surface is observed; for example, when the fluorescent dye is FITC, green dotted fluorescence is observed, and when the fluorescent dye is Rhodamine B, red fluorescence is observed. If a fluorescence signal is emitted, it indicates that the stained spores have entered the interior of the packaging material, the seal is inadequate, and it is difficult to resist microbial invasion. The filter membrane is then cultured to observe bacterial growth. If no fluorescence signal is detected, and no contaminating indicator bacteria are detected during filter membrane culture, it indicates that the packaging material has good sealing properties and can resist microbial invasion. This embodiment uses fluorescent labeling to make contamination monitoring more visual and improves the ability to identify contamination pathways.

[0061] In some embodiments, the blue light and the filter membrane are irradiated at a 45° angle, making it easier to observe the fluorescence signal.

[0062] In some embodiments, the method for testing microbial intrusion into packaging materials further includes a method validation step. Steps S10 to S50 described above are used as the experimental group. A negative control group and a positive control group are set up in reference to the experimental group. The difference is that in step S30, the negative control group does not inject indicator bacterial spore suspension into the sealed area of ​​the packaging material under test, but only simulates transportation. In contrast, the positive control group first injects indicator bacterial spore suspension into the liquid before sealing the sealed area. If no colonies appear in the experimental group, it proves that the sealing structure effectively blocks biological intrusion. If marked colonies appear, it indicates a risk of contamination at the seal. The absence of colonies in the negative control group and the presence of colonies in the positive control group verify the stability and reliability of the experimental system.

[0063] In some embodiments, the number of samples in the experimental group, negative control group, and positive control group is no less than 5 groups.

[0064] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following specific embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0065] Example 1

[0066] like Figure 1 As shown in the figure, this embodiment provides a method for testing microbial intrusion into packaging materials, and the specific steps are as follows:

[0067] S10. *Bacillus thermoamylovorans*, with an optimal growth temperature of 55℃, was selected as the contamination indicator bacterium. This strain was cultured at 55℃ to the late logarithmic phase, then subjected to heat shock at 80℃ and induced to form spores under restricted nutrient conditions. The spore pellet was then collected by centrifugation and resuspended in PBS buffer to a final concentration of 10 μL. 7 A suspension of indicator bacterial spores was obtained at CFU / mL. To achieve visual tracking of the contamination pathway, the outer wall of the spores was stained with the fluorescent dye FITC to obtain a fluorescent indicator bacterial spore suspension. The specific staining method was as follows: the spore suspension was reacted with 0.01% FITC at room temperature in the dark for 30 min. After the reaction, the spores were washed 2-3 times with PBS to remove free dye, resulting in the fluorescent indicator bacterial spore suspension.

[0068] S20. Select a 12.8L bottle filled with sterile purified water as the test sample, and place the bottle in a clean operating area for 48 hours to eliminate environmental interference bacteria.

[0069] S30. Using a sterile syringe, inject 1 mL of fluorescent indicator bacterial spore suspension into the junction of the bottle cap and the bottle mouth edge, and then immediately heat-seal the bottle mouth to complete the sealing of the bottle mouth.

[0070] S40. The sealed samples were transported in a transport vehicle for 72 hours under normal temperature conditions.

[0071] S50. After the transportation simulation, the bottle cap surface was disinfected with 75% alcohol in the laminar flow hood, and a 200mL liquid sample was extracted from the bottle by piercing the center of the cap with a sterile sampling needle. The liquid sample was then filtered through a 0.22μm filter membrane, which was used for contamination monitoring and culture observation. For contamination detection, the filter membrane was placed on a clean glass slide and illuminated at a 45° angle with blue light (excitation wavelength 488nm) or a green flashlight in the dark to observe whether the filter membrane showed a green tint. If a fluorescence signal was present, it indicated that the contamination indicator bacteria had invaded the bottle, suggesting microbial leakage in the sealing structure. The filter membrane was then laid flat on a TSA agar plate and incubated at 55°C for 48 hours to observe whether colonies grew and to further confirm whether the colonies originated from the inoculation indicator bacteria.

[0072] Method Validation

[0073] In this embodiment, the operations of steps S10 to S50 above are used as the experimental group, and a negative control group and a positive control group are set up. The negative control group and the positive control group follow the same operation as the experimental group, except that: the negative control group does not inoculate the sealed part with fluorescent indicator bacterial spore suspension, while the positive control group first inoculates the liquid with fluorescent indicator bacterial spore suspension and then seals it.

[0074] Five groups were designed for each of the above experimental groups, negative control groups, and positive control groups.

[0075] The experimental results of method validation showed that the negative controls showed no fluorescence or colonies, while all positive controls showed fluorescence signals and colony growth. The microbial blocking performance of the sealing structure in the experimental group was determined based on the presence or absence of fluorescence or positive culture results. If the experimental group samples showed no fluorescence signal or colony growth, it indicated that the sealing structure was well-sealed and could effectively prevent the intrusion of spore-forming contaminants; if fluorescence signals or colony growth were present, it suggested a risk of contamination. In this example, the experimental group showed no fluorescence signal or colony growth, indicating that the packaging material under test had good sealing performance.

[0076] Example 2

[0077] Example 2 was tested using the same method as Example 1. The difference was that Example 2 did not use fluorescent dyes and did not observe fluorescence signals. Instead, the filter membrane was directly cultured and the colonies were observed.

[0078] Example 2, following the method described in Example 1, yielded the following results: No colonies appeared in the negative controls, while colony growth was detected in all positive controls. For the experimental group, the microbial blocking performance of the sealing structure was determined based on the presence or absence of positive culture results. If no colonies grew in the experimental group samples, it indicates good sealing performance, effectively preventing the intrusion of spore-forming contaminants; if colonies grew, it suggests a risk of contamination. In this example, the absence of colony growth in the experimental group indicates that the tested packaging material has good sealing performance and can effectively prevent microbial invasion.

[0079] Example 3

[0080] Example 3 was tested using the same method as in Example 1, except that the fluorescent dye used in Example 3 was Rhodamine B.

[0081] Example 3, following the method described in Example 1, yielded the following results: Negative controls showed no fluorescence or colonies, while all positive controls showed fluorescence signals and colony growth. For the experimental group, the microbial blocking performance of the sealing structure was assessed based on the presence or absence of fluorescence or positive culture results. If the experimental group samples showed no fluorescence signal or colony growth, it indicated good sealing performance, effectively preventing the intrusion of spore-forming contaminants; the presence of fluorescence signals or colony growth suggested a risk of contamination. In this example, the experimental group showed no fluorescence signal or colony growth, indicating good sealing performance of the tested packaging material.

[0082] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for testing microbial intrusion in packaging materials, characterized in that, Includes the following steps: S10. Identify the contamination indicator bacteria and prepare an indicator bacteria spore suspension from the contamination indicator bacteria; S20. Fill the packaging material to be tested with sterile liquid; S30. Inject the indicator bacteria spore suspension into the sealed part of the packaging material to be tested, and then seal the sealed part. S40. Conduct transportation simulation on the sealed packaging materials to be tested; S50. Extract the liquid from the inside of the test packaging material after the transportation simulation is completed, filter and culture the extracted liquid, and detect the presence of the contamination indicator bacteria to evaluate the airtightness of the test packaging material.

2. The method for testing microbial intrusion in packaging materials according to claim 1, characterized in that, The contamination indicator bacteria include Bacillus thermophilus.

3. The method for testing microbial intrusion in packaging materials according to claim 1, characterized in that, The colony-forming unit of the indicator bacterial spore suspension is 10-1. 7 CFU / mL.

4. The method for testing microbial intrusion into packaging materials according to claim 1, characterized in that, The spores of the indicator bacteria spore suspension were labeled with a fluorescent dye.

5. The method for testing microbial intrusion into packaging materials according to claim 1, characterized in that, The fluorescent dyes include fluorescein isothiocyanate or rhodamine B.

6. The method for testing microbial intrusion in packaging materials according to claim 1, characterized in that, The sealing part is the junction between the bottle cap and the edge of the bottle mouth.

7. The method for testing microbial intrusion in packaging materials according to claim 1, characterized in that, The transportation simulation includes: placing the sealed packaging material to be tested on a transport vehicle and transporting it for 70 to 75 hours at a transport temperature of 20°C to 30°C.

8. The method for testing microbial intrusion in packaging materials according to claim 1, characterized in that, The step of extracting the liquid includes: piercing the sealed portion of the packaging material to be tested with a sterile sampling needle and extracting the liquid inside the packaging material to be tested.

9. The method for testing microbial intrusion in packaging materials according to claim 1, characterized in that, The detection of the presence of contamination indicator bacteria includes the following steps: passing the extracted liquid through a 0.22 μm filter membrane, placing the filter membrane in a culture medium for incubation, and observing whether the contamination indicator bacteria appear.

10. The method for testing microbial intrusion in packaging materials according to claim 3, characterized in that, The extracted liquid is passed through a 0.22 μm filter membrane, and the appearance of a fluorescent signal on the filter membrane is observed. The filter membrane is then placed in a culture medium for incubation, and the presence of the contamination indicator bacteria is observed.

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