Method for identifying fungal spore invasion of packing material and application of method

Abstract

The invention discloses a method for identifying fungal spore invasion of a packing material and application of the method, and belongs to the technical field of packing material quality detection. The method comprises the following steps: adding a sterile culture medium into a sterile packing material, and sealing the packing material; soaking the packing material containing the culture medium in a physiological saline solution containing fungal spores, so that the exterior of the packing material containing the culture medium is in direct contact with the fungal spores; placing the soaked packing material in a culture environment for culture; detecting whether fungi grow in a culture medium in the cultured packing material or not, and if fungi are detected in the culture medium, judging that fungal spores invade the packing material; and if no fungus is detected from the culture medium, judging that the fungal spores do not invade the packing material. According to the identification method, the invasion process of fungal spores can be simulated, the accuracy and reliability of a detection result are improved, the invasion resistance effect of the packaging material on various fungi can be identified at the same time, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of packaging material quality detection, and in particular to a method for identifying fungal spore invasion into packaging materials. Background Art

[0002] In the prior art, the detection of fungal invasion by packaging materials mainly relies on the following methods: (1) Fungal challenge test: by exposing the packaging material to a specific fungal environment, observe whether it can effectively block fungi. The disadvantages of this method are that the test conditions are complicated, the operation is difficult and the cost is high. (2) Gas permeability test: This method evaluates the gas permeability of the packaging material and indirectly infers the ability of fungi to invade. However, this method cannot directly measure fungal invasion and is often not targeted. (3) Physical penetration test: by applying a certain pressure, the physical strength of the packaging material is evaluated to determine its ability to protect against fungi. However, this method cannot simulate the actual fungal invasion process, and the test results may not be accurate enough.

[0003] The above existing methods generally have the following defects: the testing process is complicated, the operation is difficult, the testing cycle is long and the cost is high, and it is often difficult to accurately simulate the actual invasion process of fungi, thus affecting the accuracy and reliability of the test results. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for identifying fungal spore invasion of packaging materials, so as to solve the technical problems in the fungal invasion detection process of packaging materials, such as complex testing process, great difficulty in operation, long testing cycle, high cost, difficulty in simulating the actual fungal invasion process, thus affecting the accuracy and reliability of the detection results, and generally only a single bacterial test can be performed.

[0005] To achieve the above object, the present invention provides a method for identifying fungal spores invading packaging materials, characterized in that it comprises the following steps:

[0006] Adding a sterile culture medium into a sterile packaging material and sealing the packaging material;

[0007] Soaking a packaging material containing a culture medium in a physiological saline solution containing fungal spores, so that the outside of the packaging material containing the culture medium directly contacts the fungal spores;

[0008] placing the soaked packaging material in a culture environment for culture;

[0009] The culture medium inside the packaging material after culture is detected to determine whether there is fungal growth. If fungi are detected from the culture medium, it is determined that the fungal spores have invaded the packaging material; if fungi are not detected from the culture medium, it is determined that the fungal spores have not invaded the packaging material.

[0010] In some embodiments of the present invention, the packaging material comprises a plastic packaging material.

[0011] In some embodiments of the present invention, the culture medium comprises PDB medium.

[0012] In some embodiments of the present invention, the content of the culture medium inside the packaging material is 300ml-500ml.

[0013] In some embodiments of the present invention, the concentration of the fungal spores in the physiological saline solution is 10^3 CFU / mL.

[0014] In some embodiments of the present invention, the fungal spores include Ceratocystis spores and / or Aspergillus versicolor spores.

[0015] In some embodiments of the present invention, in the step of immersing the packaging material containing the culture medium in a physiological saline solution containing fungal spores, the immersion time is 24 hours to 48 hours.

[0016] In some embodiments of the present invention, in the step of placing the soaked packaging material in a culture environment for culture, the culture temperature is 28° C.; and / or the culture time is 5 to 10 days.

[0017] In some embodiments of the present invention, in the step of detecting whether fungi grow in the culture medium inside the packaging material after culture, changes in turbidity and OD value of the culture medium are recorded; and / or, whether fungi are present in the culture medium is observed under a microscope.

[0018] The present invention also provides an application of the above-mentioned method for identifying fungal spore invasion of packaging materials in the field of food.

[0019] The beneficial effects that can be achieved by the present invention are:

[0020] The present invention adds a sterile culture medium into a sterile packaging material and seals the packaging material to keep the interior of the packaging material sterile, then immerses the sealed packaging material in a physiological saline solution containing fungal spores so that the exterior of the packaging material directly contacts the fungal spores, then takes out the packaging material and places it in a culture environment to culture the fungal spores attached to the packaging material, and after the culture is completed, determines whether the culture medium inside the packaging material is contaminated by fungi, if fungi are detected, it means that the fungal spores invade the interior of the packaging material from the exterior of the packaging material, and if no fungi are detected, it means that the fungal spores do not invade the interior of the packaging material, and the packaging material can block the invasion of fungi and has good quality.

[0021] The present invention utilizes physiological saline containing fungal spores to simulate an actual biological invasion environment, which is more representative than a single bacterial test in the prior art, can more realistically simulate the invasion process of fungal spores, improve the accuracy and reliability of the test results, and can simultaneously identify the anti-invasion effects of packaging materials on multiple fungi, thereby improving the efficiency of the test. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0023] Figure 1 The present invention is a schematic diagram of a process for identifying the invasion of fungal spores into packaging materials.

[0024] Figure 2 The figure is a comparison of the turbidity of the culture medium in the packaging material of Example 1 of the present invention and the control group 1.

[0025] Figure 3 This is a microscopic image of the culture medium in the packaging material of the control group 1 of the present invention.

[0026] Figure 4 This is a microscopic image of the culture medium in the packaging material of Example 1 of the present invention.

[0027] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0028] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] In the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0031] The present invention provides a method for identifying fungal spore invasion of packaging materials, comprising the following steps:

[0032] S10, adding a sterile culture medium into the sterile packaging material and sealing the packaging material;

[0033] S20, soaking the packaging material containing the culture medium in a physiological saline solution containing fungal spores, so that the outside of the packaging material containing the culture medium directly contacts the fungal spores;

[0034] S30, placing the soaked packaging material in a culture environment for culture;

[0035] S40, detecting whether there is fungus growth in the culture medium inside the packaging material after culture. If fungus is detected from the culture medium, it is determined that the fungal spores have invaded the packaging material; if fungus is not detected from the culture medium, it is determined that the fungal spores have not invaded the packaging material.

[0036] The present invention adds a sterile culture medium into a sterile packaging material and seals the packaging material to keep the interior of the packaging material sterile, then immerses the sealed packaging material in a physiological saline solution containing fungal spores so that the exterior of the packaging material directly contacts the fungal spores, then takes out the packaging material and places it in a culture environment to culture the fungal spores attached to the packaging material, and after the culture is completed, determines whether the culture medium inside the packaging material is contaminated by fungi, if fungi are detected, it means that the fungal spores invade the interior of the packaging material from the exterior of the packaging material, and if no fungi are detected, it means that the fungal spores do not invade the interior of the packaging material, and the packaging material can block the invasion of fungi and has good quality.

[0037] The present invention utilizes physiological saline containing fungal spores to simulate an actual biological invasion environment, which is more representative than a single bacterial test in the prior art, can more realistically simulate the invasion process of fungal spores, improve the accuracy and reliability of the test results, and can simultaneously identify the anti-invasion effects of packaging materials on multiple fungi, thereby improving the efficiency of the test.

[0038] The identification method of the present invention is applicable to a type of packaging material that can be sealed and is not easily soluble in aqueous solution. In some embodiments, the packaging material includes plastic packaging material, such as plastic bottle packaging material for bottled beverages. Therefore, the identification method of the present invention is applicable to the food field.

[0039] In step S10, before adding the culture medium inside the packaging material, it is necessary to ensure that the packaging material is sterile, and the sterile range includes the inside of the packaging material. At the same time, the culture medium must be sterilized to prevent the inside of the packaging material from being contaminated by the culture medium containing fungi before adding the culture medium and affecting the accuracy of the test. Similarly, sealing after adding the culture medium is also to avoid being contaminated by fungi in the environment before the test and affecting the accuracy of the test.

[0040] In some embodiments, the culture medium includes PDB culture medium, i.e. potato dextrose broth culture medium. PDB culture medium contains potato extract and glucose, which can provide microorganisms with sufficient carbon source, nitrogen source, vitamins and other nutrients required for growth. Since PDB culture medium is rich in nutrients, it can support the growth of most fungi and can support the identification and detection of different fungi invading packaging materials. Moreover, the preparation method of PDB culture medium is relatively simple. Usually, it is only necessary to mix the culture medium powder with water, boil it to dissolve it, and then sterilize it. In general, PDB culture medium has the advantages of rich nutrition, easy preparation, and wide application. It is widely used in microbiological research and applications, and is suitable for the detection, separation and cultivation of various fungi.

[0041] In the present invention, the amount of culture medium added to the packaging material can be determined according to the capacity of the packaging material, so that the packaging material with the culture medium added is easy to seal and not easy to cause the packaging material to rupture. In some embodiments, the amount of culture medium added to the packaging material is 300ml to 500ml.

[0042] In step S20, a physiological saline solution containing fungal spores is prepared, and the packaging material containing the culture medium is immersed in the physiological saline solution so that the outside of the packaging material is in direct contact with the fungal spores in the physiological saline solution. This is to simulate the actual biological invasion environment, which can not only enhance the reliability and accuracy of the detection, but also speed up the detection process.

[0043] In the present invention, the entire sealing packaging material can be immersed in a physiological saline solution to detect the overall protection ability of the packaging material against fungi, and a partial area of ​​the packaging material can be immersed in a physiological saline solution to detect the protection ability of the area against bacteria. For example, the sealing part of the packaging material can be immersed in a physiological saline solution to detect the sealing effect of the packaging material.

[0044] In the present invention, the type of fungal spores can be selected according to the items to be detected.

[0045] For example, in the field of food, especially in the field of beverages, multiple species in the genus Talaromyces, Ascomycetes, Euroticulariales, Trichodermaceae, have been reported to produce heat-resistant spores, which can cause pasteurized juice and fruit products to spoil and produce mycotoxins in food. Therefore, Talaromyces has a special status in the food industry. For example, Talaromyces verruculosus in the genus Talaromyces is often detected in pasteurized juice, dairy products and soft drinks, which will destroy the quality of juice, dairy products and soft drinks, and is an important indicator for detecting food quality and food packaging quality.

[0046] For example, Aspergillus versicolor in the Ascomycota, Eurotiomycetes, Eurotiomycetes, Trichodermaceae, Aspergillus genus is commonly found in humid indoor environments and foods, and often appears in dusty and water-damaged building materials such as wall panels, insulation materials, textiles, ceiling tiles, and artificial wood. Its optimal growth temperature is 22°C to 26°C, and it can also grow in the range of 4°C to 40°C. It can survive in NaCl solutions with a mass concentration of up to 30% or sucrose solutions with a mass concentration of up to 40%. It is not only an indicator of moisture problems in buildings, but also an important indicator for detecting the quality of food packaging materials.

[0047] Therefore, in some embodiments, if it is necessary to detect whether the genus Cyanobacteria is easy to invade the packaging material and to identify whether the packaging material has a good barrier effect and sealing effect on the genus Cyanobacteria, spores of the genus Cyanobacteria can be added to the physiological saline solution. If it is necessary to detect whether the genus Aspergillus is easy to invade the packaging material and to identify whether the packaging material has a good barrier effect and sealing effect on the genus Aspergillus, fungal spores of Aspergillus versicolor can be added to the physiological saline solution. In addition, fungal spores of different types can also be added to the physiological saline solution to identify the barrier effect of the packaging material on different types of fungal spores.

[0048] In some embodiments, the concentration of fungal spores in the physiological saline solution is 10^3 CFU / mL. The concentration of fungal spores in the physiological saline solution is within the above range, which can better simulate the actual biological invasion environment, can more realistically simulate the invasion process of fungal spores, and improve the accuracy and reliability of the detection results.

[0049] In some embodiments, the packaging material containing the culture medium is immersed in the physiological saline solution containing fungal spores for 24 to 48 hours. For example, it can be 24 hours, 30 hours, 48 ​​hours, etc. Under the above immersion time conditions, the actual biological invasion environment can be better simulated, the accuracy and reliability of the detection results can be improved, and the quality of the packaging material can be better reflected.

[0050] In step S30, the soaked packaging material is cultured, which can better simulate the actual biological invasion environment, speed up the detection, and enhance the reliability and accuracy of the detection.

[0051] In the present invention, the entire packaging material can be placed in an environment suitable for fungal reproduction for cultivation, so that the fungal spores adhering to the outside of the packaging material can grow and reproduce, which is convenient for detection and enhances the reliability and accuracy of the detection. Moreover, testing after cultivation is more convincing in proving the quality of the packaging material.

[0052] In some embodiments, the culture temperature for culturing the soaked packaging material is 28° C., which is a temperature condition suitable for the survival and reproduction of fungi. It can better simulate the actual biological invasion environment, speed up the detection process, and enhance the reliability and accuracy of the detection.

[0053] In some embodiments, the culturing time for the soaked packaging material is 5 to 10 days, which can be 5 days, 7 days, 10 days, etc. When the culturing period reaches a certain length, the fungi can multiply to a certain number, which is not only easy to observe and identify, but also enhances the reliability and accuracy of the detection, and is more convincing in proving the quality of the packaging material.

[0054] In step S40, the culture medium inside the cultured packaging material is detected to determine whether fungi grow therein, which can be used to determine whether fungal spores adhering to the outside of the packaging material can penetrate the packaging material and invade the inside of the packaging material. In this step, common detection methods in the art can be used to detect whether fungi grow therein.

[0055] In some embodiments, whether fungi grow in the culture medium can be determined by recording changes in turbidity and OD value of the culture medium.

[0056] In the present invention, the process of judging whether fungal spores invade the packaging material by recording the turbidity change of the culture medium is as follows: by naked eye observation, if floccules appear in the culture medium inside the packaging material, it can be judged that the fungal spores have invaded the packaging material. Further, the method of identifying the invasion of fungal spores in the packaging material of the present invention can also be used as an experimental group, and a control group can be added at the same time. The control group is designed with reference to the experimental group, but the difference is that after the control group adds a sterile culture medium inside the sterile packaging material, the packaging material is not sealed. For example, if the packaging material is a plastic bottle, the sealed lid of the plastic bottle can be unscrewed and slowly closed, with the goal of not flowing out the liquid in the bottle. After adding the control group, the turbidity of the culture medium in the experimental group and the control group can be compared to judge whether fungal growth occurs in the experimental group. For example, if the culture medium of the control group is turbid, the culture medium of the experimental group is also turbid, which is judged to be poor in the ability of the packaging material to resist fungal spores. If the culture medium of the experimental group is not turbid, it is judged that the ability of the packaging material to resist fungal spores is better.

[0057] In the present invention, the process of determining whether fungal spores have invaded the packaging material by recording the OD value of the culture medium is as follows: the method of identifying the invasion of fungal spores into the packaging material of the present invention is used as the experimental group, and a control group is added at the same time. The control group is designed with reference to the experimental group, but the difference is that after the control group adds the sterile culture medium inside the sterile packaging material, the packaging material is not sealed. For example, if the packaging material is a plastic bottle, the sealed lid of the plastic bottle can be unscrewed and then slowly closed, with the goal of preventing the liquid in the bottle from flowing out. After the control group is added, the OD value of the culture medium at 550nm inside the packaging material of the control group is set to 0, and the OD value of the experimental group is measured. If the OD value of the experimental group is greater than or equal to 0.2, it can be determined that the fungal spores have invaded the packaging material.

[0058] In some embodiments, the culture medium can also be observed under a microscope to see if there is fungal growth.

[0059] In step S40, when testing the culture medium inside the packaging material, it is necessary to avoid contamination by fungi outside the packaging material that may cause errors in the test results.

[0060] The method for identifying fungal spore invasion of packaging materials of the present invention can be applied to the food field, such as plastic packaging materials used for beverages.

[0061] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0062] Example 1

[0063] The steps of the method for identifying fungal spore invasion of packaging materials in this embodiment are as follows:

[0064] S10: Select a plastic package with a capacity of 1000 ml and can be sealed by a sealing lid, sterilize the inside and outside of the plastic package, and then inject 300 ml of sterilized PDB culture medium into the interior of the package under a sterile environment and seal the plastic package by the sealing lid.

[0065] S20: Prepare a physiological saline solution containing 10^3 CFU / mL of Talaromyces verrucosus spores, and soak the packaging material injected with PDB culture medium in the physiological saline solution for 24 hours to ensure that the outside of the packaging material is in direct contact with the physiological saline solution so that the fungal spores can adhere to the outside of the packaging material.

[0066] S30: Take out the packaging material from step S20, and place the packaging material in a culture environment at 28° C. for 7 days.

[0067] S40: Visually observe the turbidity of the culture medium inside the packaging material after the culture in step S30, measure the change in the OD value of the culture medium, and observe with a microscope to see whether there is fungal growth in the culture medium.

[0068] Example 2

[0069] Example 2 The same type of packaging material was identified by referring to the identification method of Example 1, except that the fungal spores used were Aspergillus versicolor spores.

[0070] Example 3

[0071] Example 3 The same type of packaging material was identified by referring to the identification method of Example 1, except that the physiological saline contained 10^3 CFU / mL of Talaromyces verrucosus spores and 10^3 CFU / mL of Aspergillus versicolor spores.

[0072] Control group 1

[0073] Comparative Example 1 The same type of packaging material is identified by referring to the identification method of Example 1, except that in step S10, the sterile packaging material is not completely sealed. The specific operation is: unscrewing the sealed lid of the packaging material and then slowly closing it to prevent liquid from leaking out.

[0074] Control group 2

[0075] Comparative Example 2 refers to the identification method of Example 2 to identify the same type of packaging materials. The difference is that in step S10, the sterile packaging material is not completely sealed. The specific operation is: unscrew the sealed lid of the packaging material and then slowly close it to prevent liquid from leaking out.

[0076] Control group 3

[0077] Comparative Example 3 identifies the same type of packaging material with reference to the identification method of Example 3, except that in step S10, the sterile packaging material is not completely sealed. The specific operation is: unscrewing the sealed lid of the packaging material and then slowly closing it to prevent liquid from leaking out.

[0078] The specific operation and results of step S40 of Example 1 and Control Group 1 are as follows:

[0079] 1. Observe the turbidity of the culture medium inside the packaging material of Comparative Example 1 and Control Group 1. The results are shown in Figure 2 , Figure 2 The circled ones are control group 1, and the ones without circled ones are example 1. The observed conditions are recorded in Table 1. Figure 2It can be seen that no flocs appeared in the culture medium of Example 1, but flocs appeared in the culture medium of Control Group 1, indicating that the culture medium in Control Group 1 where the bottle cap was not tightened was contaminated by fungi, while the culture medium in Example 1 after sealing treatment was not contaminated by fungi, and the packaging material has a better effect in resisting fungal invasion.

[0080] 2. The OD value of the culture medium at 550 nm inside the packaging material of the control group 1 was set to 0, and the OD value of the culture medium at 550 nm inside the packaging material of Example 1 was measured and recorded in Table 1.

[0081] 3. Observe the culture medium inside the packaging materials of Example 1 and Control Group 1 under a microscope to see if there are fungi. Figure 3 As shown, fungi appeared, and the culture medium of Example 1 was as follows Figure 4 As shown, no fungi were present.

[0082] The operation of step S40 of Example 2 and Control Group 2 and Example 3 and Control Group 3 specifically refers to the operation of Example 1 and Control Group 1, and the turbidity and OD value of the culture medium of Example 2 and Control Group 2 and Example 3 and Control Group 3 are observed and measured in turn.

[0083] Table 1

[0084] Experimental Group Turbidity OD value Example 1 No flocs 0.125 Control group 1 White flocculent 0.432 Example 2 No flocs 0.114 Control group 2 Floccules 0.374 Example 3 No flocs 0.096 Control group 3 Floccules 0.503

[0085] The packaging materials of Examples 1 to 3 have good resistance to fungal spores, and no fungal spore invasion occurs. However, the control groups of Examples 1 to 3, i.e., Control Groups 1 to 3, did not tighten the bottle caps and did not seal, and fungal spore invasion occurred. It can be seen that the present invention uses physiological saline containing fungal spores to simulate the actual biological invasion environment, which is more representative than the single bacterial test in the prior art, can more realistically simulate the invasion process of fungal spores, improve the accuracy and reliability of the test results, and can simultaneously identify the anti-invasion effect of the packaging material on multiple fungi, thereby improving the efficiency of the test.

[0086] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for identifying fungal spore invasion of packaging materials, characterized in that: The following steps are involved: Adding a sterile culture medium into a sterile packaging material and sealing the packaging material; Soaking a packaging material containing a culture medium in a physiological saline solution containing fungal spores, so that the outside of the packaging material containing the culture medium directly contacts the fungal spores; placing the soaked packaging material in a culture environment for culture; The culture medium inside the packaging material after culture is detected to determine whether there is fungal growth. If fungi are detected from the culture medium, it is determined that the fungal spores have invaded the packaging material; if fungi are not detected from the culture medium, it is determined that the fungal spores have not invaded the packaging material.

2. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: The packaging material includes plastic packaging material.

3. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: The culture medium includes PDB medium.

4. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: The content of the culture medium inside the packaging material is 300ml to 500ml.

5. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: The concentration of the fungal spores in the physiological saline solution is 10^3 CFU / mL.

6. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: The fungal spores include Ceratocystis spores and / or Aspergillus versicolor spores.

7. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: In the step of immersing the packaging material containing the culture medium in the physiological saline solution containing fungal spores, the immersion time is 24 hours to 48 hours.

8. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: In the step of placing the soaked packaging material in a culture environment for culture, the culture temperature is 28° C.; and / or the culture time is 5 to 10 days.

9. The method for identifying fungal spore invasion of packaging materials according to claim 1, characterized in that: In the step of detecting whether fungi grow in the culture medium inside the packaging material after culture, changes in turbidity and OD value of the culture medium are recorded; and / or, whether fungi grow in the culture medium is observed under a microscope.

10. Use of the method for identifying fungal spore invasion of packaging materials according to any one of claims 1 to 9 in the food field.

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