Aspergillus braziliensis spore storage method and application thereof

Abstract

The invention relates to the technical field of microorganism reproduction and preservation, in particular to a storage method and application of aspergillus braziliensis spores. According to the method, a specific nutrient solution is used for preparing an Aspergillus Brazilii spore suspension, the Aspergillus Brazilii spore suspension is applied to the surface of a crosslinked polyethylene material, and after drying treatment, Aspergillus Brazilii spores are attached to the surface of the crosslinked polyethylene material in a dormant state; and the aspergillus braziliensis spores can be stably stored for more than 10 months in a normal-temperature and normal-pressure closed environment. The problem that microorganisms are prone to inactivation due to the fact that the time span of microorganism sample preparation and on-orbit starting is large in a space station test is directly solved, the effectiveness of the test is ensured, and waste of precious on-orbit resources is avoided. Dependence on ascending and on-orbit low-temperature storage equipment is completely eliminated, precious and tense low-temperature storage resources of a space station are greatly saved, and a large-scale and long-period space microbial corrosion test can be possibly carried out.

Description

Technical Field

[0001] This invention relates to the field of microbial propagation and preservation technology, and in particular to a method for storing Aspergillus brasiliensis spores and its application. Background Technology

[0002] While providing a safe haven for astronauts' long-term stays, the space station also creates favorable conditions for microbial growth. The microgravity of space can alter the biological characteristics of microorganisms, leading to a series of adaptive changes such as accelerated growth rates, enhanced metabolic activity, and improved biofilm formation capabilities. These changes could potentially further exacerbate the erosion and corrosion of spacecraft materials by microorganisms. Previously, the International Space Station has repeatedly detected biofilms formed by microorganisms such as Penicillium and Aspergillus corroding critical spacecraft materials, causing equipment failures and affecting the long-term stable operation of the space station. Therefore, research on the microbial corrosion of materials under space conditions is of great significance. This research is not only a crucial foundation for accurately assessing the long-term reliability of materials in orbit and ensuring the safe and stable operation of the space station, but also provides indispensable scientific basis and data support for the future optimization and lifespan prediction of space station materials.

[0003] Aspergillus brasiliensis ( Aspergillus brasiliensis Aspergillus brasiliensis belongs to the genus Aspergillus and can be used in fields such as verifying preservative efficacy, quality control of culture media, and industrial production of lactic acid. Conducting Aspergillus brasiliensis experiments on the space station is of great significance; however, a key challenge in conducting Aspergillus brasiliensis experimental research on the space station lies in the long-term storage of Aspergillus brasiliensis samples, specifically in the following two aspects: First, the experiment has a long cycle and requires high levels of viability maintenance for Aspergillus brasiliensis. The time span from the preparation of microbial samples on the ground to the start of the experiment in orbit can be as long as 6 months or more. This places extremely high demands on the long-term survival ability of Aspergillus brasiliensis, and it is necessary to avoid wasting valuable space station resources due to its inactivation.

[0004] Second, there is a limitation of cryogenic storage resources. Currently, commonly used methods for microbial preservation on Earth (such as slant cryopreservation and liquid nitrogen cryopreservation) all rely on dedicated cryogenic equipment. However, in space station missions, resources for the cryogenic ascent of biological samples are relatively scarce, making it difficult to support the needs of large-scale or long-term cryogenic storage.

[0005] There is a lack of existing technologies for the long-term stable storage of Aspergillus brasiliensis in an environment of normal temperature and pressure, so as to conduct experiments on the space station. Summary of the Invention

[0006] This invention provides a spore storage method and its application, which solves the problem in the prior art that Aspergillus brasiliensis is easily inactivated and its storage requires low-temperature storage resources due to the large time span between sample preparation and on-orbit start-up in space station experiments.

[0007] This invention provides a method for storing Aspergillus brasiliensis spores, comprising: preparing a suspension of Aspergillus brasiliensis spores using a nutrient solution, applying the suspension of Aspergillus brasiliensis spores to the surface of a cross-linked polyethylene material, and allowing the Aspergillus brasiliensis spores to adhere to the surface of the cross-linked polyethylene material in a dormant state after drying. The 1L nutrient solution contains the following components: 0.68-0.72g KH2PO4, 0.29-0.31g K2HPO4, 0.48-0.52g MgSO4·7H2O, 1.8-2.2g NaNO3, 0.008-0.012g FeSO4·7H2O, 0.48-0.52g KCl, and 29-31g sucrose, with the balance being water. A suspension of Aspergillus brasiliensis spores was prepared using this nutrient solution. After drying, dormant Aspergillus brasiliensis was loaded onto a cross-linked polyethylene material. Even after 10 months of sealed storage at room temperature and pressure, high activity was maintained, demonstrating excellent storage performance for Aspergillus brasiliensis spores. Applying this spore storage method to space station experiments achieves long-term stable spore storage while eliminating reliance on onboard and in-orbit cryogenic storage equipment.

[0008] According to the method for storing Aspergillus brasiliensis spores of the present invention, cross-linked polyethylene material with dormant Aspergillus brasiliensis spores attached is sealed and stored.

[0009] In some preferred embodiments of the present invention, the spore-bearing material is sealed and stored in a sealed device with a leakage rate better than 5.0 × 10⁻⁶. -4 Pa·m 3 / s. In the method for storing Aspergillus brasiliensis spores of the present invention, controlling the leakage rate within this range can ensure the long-term storage effect of Aspergillus brasiliensis spores.

[0010] In some preferred embodiments of the present invention, the spore-bearing material is sealed and stored in a sealed device with a leakage rate of 1.0 × 10⁻⁶. -5 -5.0×10 -4 Pa·m 3 / s.

[0011] In some preferred embodiments of the present invention, 5.0 × 10 -5 -5.0×10 -4 Pa·m 3 / s.

[0012] In some embodiments of the present invention, the application method may be spraying or dripping.

[0013] According to the method for storing Aspergillus brasiliensis spores of the present invention, the drying conditions include: temperature control at 20℃–25℃ and air velocity control at 0.3–0.5 m / s. Using these drying conditions, the drying efficiency is high, and damage to the Aspergillus brasiliensis spores can be avoided.

[0014] Preferably, the drying process is completed in a biosafety cabinet.

[0015] In some embodiments of the present invention, the concentration of the Aspergillus brasiliensis spore suspension is 1.0 × 10⁻⁶. 6 -1.0×10 8 CFU / mL.

[0016] According to the method for storing Aspergillus brasiliensis spores of the present invention, the concentration of the Aspergillus brasiliensis spore suspension is 1.0 × 10⁻⁶. 6 -1.0×10 8 For drying, first apply 1.6-1.8 mL of Aspergillus brasiliensis spore suspension to the surface of the cross-linked polyethylene material and dry for 28-32 minutes, then apply another 1.6-1.8 mL of Aspergillus brasiliensis spore suspension to the surface of the cross-linked polyethylene material and dry for 38-42 minutes. Using this drying method, combined with the aforementioned drying temperature and air velocity, can effectively inhibit the metabolic activity of Aspergillus brasiliensis spores while avoiding damage caused by excessive dehydration, which is particularly beneficial for the long-term stable storage of Aspergillus brasiliensis spores.

[0017] In some embodiments of the present invention, the concentration of the Aspergillus brasiliensis spore suspension is 2.5 × 10⁻⁶. 7 -3.9×10 7 CFU / mL, preferably 2.5 × 10⁻⁶. 7 -3.2×10 7 CFU / mL.

[0018] In some preferred embodiments of the present invention, the application method is spraying; the spraying distance during spraying is 15-20cm.

[0019] According to the method for storing Aspergillus brasiliensis spores of the present invention, the Aspergillus brasiliensis is the standard strain ATCC16404. ATCC 16404, as a representative of filamentous fungi, is widely used as a standard strain in various production and testing processes in the pharmaceutical, food, water, and cosmetic industries. The technical solution of the present invention has excellent preservation effects for this strain.

[0020] According to the method for storing Aspergillus brasiliensis spores of the present invention, the cross-linked polyethylene material is in sheet form. In the present invention, the sheet material facilitates spore attachment.

[0021] According to the method for storing Aspergillus brasiliensis spores of the present invention, cross-linked polyethylene material with dormant Aspergillus brasiliensis spores is sealed and stored at room temperature and pressure.

[0022] According to the method for storing Aspergillus brasiliensis spores of the present invention, the ambient temperature is 20–25°C and the ambient pressure is 96.2–106.4 kPa.

[0023] The present invention also provides the application of the Brazilian Aspergillus spore storage method in space station experiments.

[0024] According to the application described in this invention, Aspergillus brasiliensis spores are attached to the surface of cross-linked polyethylene material in a dormant state on the ground.

[0025] According to the application described in this invention, the space station test is to test the degree of corrosion of cross-linked polyethylene materials by Aspergillus brasiliensis on the space station.

[0026] The present invention also provides a method for testing the degree of corrosion of materials by Aspergillus brasiliensis on a space station, comprising: the Aspergillus brasiliensis being derived from Aspergillus brasiliensis spores stored by the Aspergillus brasiliensis spore storage method.

[0027] The method for testing the corrosion degree of Aspergillus brasiliensis on materials on a space station includes using the described Aspergillus brasiliensis spore storage method, activating the stored Aspergillus brasiliensis spores by injecting water, and observing the state of the cross-linked polyethylene material. This invention's test method is simple in activating the spores; sterile water is sufficient, simplifying the test operation and reducing economic and labor costs.

[0028] Preferably, the test method of the present invention for testing the degree of corrosion of cross-linked polyethylene materials by Aspergillus brasiliensis on a space station includes the following steps: (1) Immobilization treatment of Aspergillus brasiliensis spores: The suspension of Aspergillus brasiliensis spores was uniformly applied to the surface of cross-linked polyethylene material sheet. After drying, the Aspergillus brasiliensis spores were immobilized on the surface of cross-linked polyethylene material in a dormant state, forming an integrated test unit of "Aspergillus brasiliensis spores-cross-linked polyethylene material". (2) Sealed storage at ambient temperature and pressure: The test unit shall be placed in a sealed device, and the sealing performance shall meet the requirement that the leakage rate is better than 5.0 × 10⁻⁶. -4 Pa·m 3 / s, microbial samples were stored under normal temperature and pressure and sealed conditions from ground preparation to on-orbit start-up test. Tests showed that the activity retention period of Aspergillus brasiliensis spores was no less than 10 months. (3) On-orbit water injection activation: In the space station environment, the Aspergillus brasiliensis spores are rehydrated and activated by injecting liquid into the sealed device, and the material microbial corrosion test is directly started.

[0029] The liquid may be sterile water, nutrient solution, or any other liquid that can revive fungal spores.

[0030] A further preferred embodiment of the present invention is a test method for testing the degree of corrosion of cross-linked polyethylene materials by Aspergillus brasiliensis on a space station, comprising the following steps: (1) Immobilization treatment of Aspergillus brasiliensis spores Preparation of Aspergillus brasiliensis spore suspension: Take mature spore plates cultured for 7-14 days, wash off the spores with nutrient solution (nutrient solution composition shown in Table 1), disperse by shaking, and prepare a spore suspension of uniform concentration. Adjust the concentration to 1.0 × 10⁻⁶. 6 -1.0×10 8 CFU / mL. Subsequently, the suspension was applied evenly to the surface of the material sheet by spraying or dripping.

[0031] Table 1. Nutrient Solution Composition Table Element content <![CDATA[Potassium dihydrogen phosphate (KH2PO4)]]> 0.7g <![CDATA[Dipotassium hydrogen phosphate (K2HPO4)]]> 0.3g <![CDATA[Magnesium sulfate (MgSO4·7H2O)]]> 0.5g <![CDATA[Sodium nitrate (NaNO3)]]> 2.0g <![CDATA[Ferrous sulfate (FeSO4·7H2O)]]> 0.01g Potassium chloride (KCl) 0. 5g sucrose 30.0g distilled water Up to 1000mL Surface drying: Material sheets inoculated with Aspergillus brasiliensis spore suspension were placed in a Class 100 biosafety cabinet and dried at room temperature with an air velocity controlled at 0.3-0.5 m / s for 30-60 minutes, allowing the spores to adhere firmly to the material surface in a dry dormant state. This forms an integrated experimental unit for "Aspergillus brasiliensis spores-crosslinked polyethylene material".

[0032] (2) Store in a sealed container at room temperature and pressure. The dried "Brazilian Aspergillus spores-crosslinked polyethylene" was transferred into a sealed device, which was then sealed to form a completely sealed space. The sealed culture tank was then stored and transported at room temperature (20℃–25℃) and normal pressure (101.3 kPa ±5%) before being delivered to the space launch site for on-orbit testing.

[0033] (3) Activation by in-orbit water injection At the start of the experiment, liquid was injected into the sealed device via remote command. The liquid rapidly rehydrated and activated the dried spores, causing them to germinate and grow directly on the surface of the material sheet, initiating a microbial corrosion process on the material. The entire activation process did not require opening the device or any microbial manipulation.

[0034] The present invention has the following beneficial effects: (1) Long-term, ambient temperature and pressure storage of spores is achieved: By pre-fixing spores onto the surface of materials and drying them, this invention enables spores to be stably stored in a closed environment at ambient temperature and pressure for more than 10 months. This directly solves the problem of easy inactivation of microorganisms caused by the large time span between microbial sample preparation and on-orbit start-up in space station experiments, ensuring the effectiveness of the experiment and avoiding the waste of valuable on-orbit resources.

[0035] (2) Significantly reduces dependence on scarce space station resources: This method completely eliminates the dependence on uplink and on-orbit cryogenic storage equipment, greatly saving the precious and scarce cryogenic storage resources of the space station, making it possible to carry out large-scale, long-term space microbial corrosion experiments.

[0036] (3) Greatly simplifies on-orbit operation procedures and improves safety: The "spore-material" integrated test unit created by this invention allows all test preparation to be completed on the ground. It simplifies the complex on-orbit microbial inoculation and activation process to automatic spore activation through remote command-controlled liquid injection. This method not only eliminates the need for astronauts to perform professional microbiological operations on-orbit, significantly reducing their workload, but also fundamentally eliminates the risk of microbial leakage and cross-contamination within the cabin because the entire process is completed in a sealed device. This effectively protects the health of astronauts and the safety of the space station platform, and provides a feasible technical path for the future realization of automated, high-throughput materials biology evaluation of the space station. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This is a diagram showing the activity of Aspergillus brasiliensis spores on different material surfaces after long-term storage, as described in Example 1 of this invention.

[0039] Figure 2 This is a diagram showing the activity of Aspergillus brasiliensis spores on different material surfaces after long-term storage when the sealing requirements are not met, in Embodiment 2 of the present invention.

[0040] Figure 3 This is the verification of the growth morphology and activity of Aspergillus brasiliensis on the surface of cross-linked polyethylene material after on-orbit testing in Embodiment 2 of the present invention. Figure 3 Image A shows the growth morphology of Aspergillus brasiliensis on the surface of cross-linked polyethylene material after in-orbit testing. Figure 3 Image B is a verification diagram of the activity of Aspergillus brasiliensis on the surface of cross-linked polyethylene material after in-orbit testing. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] Example 1: Ground-based simulated Aspergillus brasiliensis spore storage activity verification test This embodiment uses ground simulation tests to verify that the leakage rate of the sealed device is 2.7 × 10⁻⁶. -4 Pa·m 3 The long-term storage effect of Aspergillus brasiliensis spores using the method described in this invention under the condition of / s.

[0043] 1. Preparation of spore suspension: Aspergillus brasiliensis (… Aspergillus brasiliensis Inoculate spores (ATCC 16404) onto PDA plates and incubate at 28°C for 10 days. Add 5 mL of sterile nutrient solution (nutrient solution composition shown in Table 1) to the plate confluent with spores. Gently and repeatedly scrape the surface of the plate with a spreader to detach the spores. Collect the eluent, add glass beads, and vortex to disperse the spore clumps. Filter through sterile gauze to remove mycelium and collect the filtrate. Count the spores using a hemocytometer and adjust the spore suspension concentration to 2.8 × 10⁻⁶ using nutrient solution. 7 CFU / mL.

[0044] 2. Spore Inoculation and Fixation: Using a sterile spray bottle, evenly spray the above spore suspension from a distance of 15-20 cm above the sterile material sheet, continuously spraying 1.8 mL until a uniform and dense distribution of fine droplets appears on the surface of the material sheet. Then, place the inoculated material sheet in a Class 100 biosafety cabinet and blow it at a wind speed of 0.3-0.5 m / s for 30 minutes at room temperature to complete the initial drying and fixation. Repeat the above spray inoculation operation once, spraying 1.8 mL and then blowing it dry again for 40 minutes, finally forming an integrated "spore-material" experimental unit loaded with dried spores.

[0045] 3. Storage at ambient temperature and pressure: After drying and fixing, the "spore-material" units were transferred into a sealed culture tank and completely sealed. The leakage rate was 2.7 × 10⁻⁶. -4 Pa·m 3 / s. The entire culture tank was placed in a constant temperature incubator at 23℃±2℃ to simulate a normal temperature and pressure (standard atmospheric pressure) storage environment.

[0046] 4. Periodic Spore Viability Testing: Starting from the start of storage, three parallel samples are randomly selected monthly for viability testing. The specific method is as follows: Add 1 mL of sterile water to each sample and vortex for 1 min to resuspend the spores. Perform serial dilutions of the resulting eluent, and spread the appropriate dilution onto PDA plates, with two parallel plates for each dilution. Incubate all plates at 28℃ for 2 days, and then count the colony-forming units (CFU) on the plates.

[0047] Results and Analysis: The number of viable spores on the surface of the material sheet was calculated by colony counting and corresponding dilutions. The experimental results are shown in [the table below]. Figure 1When stored under ambient temperature and pressure for up to 6 months, Aspergillus brasiliensis spores loaded on the surfaces of polyacrylamide zwitterionic coatings, polyurethane coatings, cross-linked polyethylene, stainless steel, and titanium alloys remained highly active. Extended monitoring to 10 months, viable spores with resuscitation capability were continuously detected on the surfaces of all tested materials, but cross-linked polyethylene showed the best preservation effect for Aspergillus brasiliensis spores. This series of data demonstrates that the method described in this invention can provide a reliable long-term spore storage solution for microbial corrosion testing of space station materials, ensuring that spores maintain resuscitation activity throughout the storage period.

[0048] Example 2: Ground-based simulated Aspergillus brasiliensis spore storage activity verification test This embodiment examines the effect of a ground-based simulation test when the leakage rate of the sealed device is 5.5 × 10⁻⁶. -3 Pa·m 3 The long-term storage activity of Aspergillus brasiliensis spores at / s was measured to verify the direct impact of the leakage rate index, a key condition of this invention, on the spore storage effect.

[0049] 1. Preparation of spore suspension: Aspergillus brasiliensis (… Aspergillus brasiliensis (ATCC 16404) was inoculated onto PDA plates and incubated at 28℃ for 10 days. 5 mL of sterile nutrient solution (nutrient solution composition shown in Table 1) was added to the confluent plate. The plate surface was gently and repeatedly scraped with a spreader to detach the spores. The eluent was collected, and glass beads were added and vortexed to disperse the spore clumps. The solution was then filtered through sterile gauze to remove mycelium, and the filtrate was collected. The filtrate was counted using a hemocytometer, and the spore suspension concentration was adjusted to 3.7 × 10⁻⁶ using nutrient solution. 7 CFU / mL.

[0050] 2. Spore Inoculation and Fixation: Using a sterile spray bottle, evenly spray the above spore suspension from a distance of 15-20 cm above the sterile material sheet, continuously spraying 1.8 mL until a uniform and dense distribution of fine droplets appears on the surface of the material sheet. Then, place the inoculated material sheet in a Class 100 biosafety cabinet and blow it at a wind speed of 0.3-0.5 m / s for 30 minutes at room temperature to complete the initial drying and fixation. Repeat the above spray inoculation operation once, spraying 1.8 mL and then blowing it dry again for 40 minutes, finally forming an integrated "spore-material" experimental unit loaded with dried spores.

[0051] 3. Storage at ambient temperature and pressure: After drying and fixing, the "spore-material" units are transferred to a sealed culture tank and completely sealed. The leakage rate is 5.5 × 10⁻⁶. -3 Pa·m 3 / s. Place it in a constant temperature incubator at 23℃±2℃ to simulate a normal temperature and pressure (standard atmospheric pressure) storage environment.

[0052] 4. Periodic Spore Viability Testing: Starting from storage, three parallel samples were randomly selected every two months for spore viability testing. The specific method was as follows: 1 mL of sterile water was added to each sample, and the mixture was vortexed for 1 min to resuspend the spores. The resulting eluent was serially diluted, and the appropriate dilution was plated onto PDA plates, with two parallel plates for each dilution. All plates were incubated at 28℃ for 2 days, and the colony-forming units (CFU) on the plates were counted.

[0053] Results and Analysis: The number of viable spores on the surface of the material sheet was calculated by colony counting and corresponding dilutions. The experimental results are shown in [the table below]. Figure 2 After four months of storage under normal temperature and pressure conditions, all Aspergillus brasiliensis spores loaded on the surfaces of aluminum alloy, magnesium alloy, circuit board, polyimide film, and cross-linked polyethylene material were inactivated. Comparative analysis of the cross-linked polyethylene material, common to Example 1, revealed that the survival status of spores on its surface was strictly controlled by the sealing performance of the sealing device. These results fully demonstrate that the leakage rate of the sealing device plays a decisive role in ensuring the long-term survival of spores.

[0054] Example 3: Activity Verification of Aspergillus brasiliensis Spores During In-orbit Storage Test Based on the successful completion of ground simulation tests, this embodiment selects the "Aspergillus brasiliensis-crosslinked polyethylene" test unit to directly evaluate the feasibility of applying the spore storage method described in this invention to the study of microbial corrosion of space station materials through on-orbit testing on the space station.

[0055] 1. Preparation of spore suspension: Aspergillus brasiliensis (… Aspergillus brasiliensis Inoculate spores (ATCC 16404) onto PDA plates and incubate at 28°C for 10 days. Add 5 mL of sterile nutrient solution (composition shown in Table 1) to the plate confluent with spores, and gently and repeatedly scrape the surface of the plate with a spreader to detach the spores; collect the eluent, add glass beads and vortex to disperse the spore clumps, then filter through sterile gauze to remove mycelium, and collect the filtrate. Count the spores using a hemocytometer, and adjust the spore suspension concentration to 3.0 × 10⁻⁶ using nutrient solution. 7 CFU / mL.

[0056] 2. Spore Inoculation and Fixation: Using a sterile spray bottle, evenly spray the above spore suspension from a distance of 15-20 cm above the sterilized cross-linked polyethylene (XLPE) sheet, continuously spraying 1.8 mL until a uniform, dense distribution of fine droplets appears on the surface of the sheet. Then, place the inoculated XLPE in a Class 100 biosafety cabinet and blow it at room temperature with an air velocity of 0.3-0.5 m / s for 30 min to complete the initial drying and fixation. Repeat the above spray inoculation procedure once, spraying 1.8 mL and then blowing it dry again for 40 min, finally forming an integrated experimental unit of "Aspergillus brasiliensis-XLPE" loaded with dried spores.

[0057] 3. Storage and Ascent at Ambient Temperature and Pressure: In November 2023, the dried and fixed experimental units were sealed in a closed incubation tank, with a leakage rate of 2.7 × 10⁻⁶. -4 Pa·m 3 / s. It was then delivered to the launch site. The sample was launched to the Chinese space station in January 2024 aboard the Tianzhou-7 cargo spacecraft, and was transported and stored under normal temperature and pressure conditions throughout the process.

[0058] 4. In-orbit test launch: On May 14, 2024, the injection system was launched via remote command control from the ground, injecting sterile water to activate Aspergillus brasiliensis spores and start a 90-day in-orbit microbial-material interaction test.

[0059] 5. Sample Recovery and Activity Verification: After the on-orbit test, the test unit was recovered during the descent of the Shenzhou-18 manned spacecraft's return capsule. The surface of the cross-linked polyethylene material was tested for Aspergillus brasiliensis (Brazilian Aspergillus). Aspergillus brasiliensis The growth of the spores was observed through macroscopic morphological photography, and spores were scraped from the surface of the material and streaked onto PDA plates to verify their resuscitation activity. The results are as follows: Figure 2 As shown. Figure 3 As shown in Figure A, a large number of black fungal colonies are visible on the surface of the material, indicating that Aspergillus brasiliensis is active and can germinate and grow successfully. Figure 3 Figure B shows the results of streak plating, which reveals typical Aspergillus brasiliensis colonies with a characteristic yellowish-brown hue, typical morphology and structure, well-developed hyphae and abundant spore masses. This indicates that the Aspergillus brasiliensis spores obtained from the material surface have good recovery activity, can complete their life cycle, and possess normal metabolic and reproductive capabilities.

[0060] 6. Observation of the corrosion degree of cross-linked polyethylene material: Remove Aspergillus brasiliensis and observe the corrosion degree of cross-linked polyethylene material.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for storing Aspergillus brasiliensis spores, characterized in that, include: A suspension of Aspergillus brasiliensis spores was prepared using a nutrient solution. The suspension was then applied to the surface of a cross-linked polyethylene material. After drying, the Aspergillus brasiliensis spores were allowed to adhere to the surface of the cross-linked polyethylene material in a dormant state. The 1L nutrient solution contains the following components: 0.68-0.72g KH2PO4, 0.29-0.31g K2HPO4, 0.48-0.52g MgSO4·7H2O, 1.8-2.2g NaNO3, 0.008-0.012g FeSO4·7H2O, 0.48-0.52g KCl, and 29-31g sucrose, with the balance being water.

2. The method for storing Aspergillus brasiliensis spores according to claim 1, characterized in that, The spore-loaded material was sealed and stored in a closed system with a leakage rate better than 5.0 × 10⁻⁶. -4 Pa·m 3 / s.

3. The method for storing Aspergillus brasiliensis spores according to any one of claims 1-2, characterized in that, The drying conditions include: temperature control at 20℃–25℃ and wind speed control at 0.3–0.5 m / s.

4. The method for storing Aspergillus brasiliensis spores according to claim 3, characterized in that, The concentration of the Aspergillus brasiliensis spore suspension was 1.0 × 10⁻⁶. 6 -1.0×10 8 CFU / mL, first apply 1.6-1.8 mL of Aspergillus brasiliensis spore suspension to the surface of the cross-linked polyethylene material and dry for 28-32 min, then apply 1.6-1.8 mL of Aspergillus brasiliensis spore suspension to the surface of the cross-linked polyethylene material and dry for 38-42 min.

5. The method for storing Aspergillus brasiliensis spores according to claim 4, characterized in that, The application method is spraying; The spraying distance during application is 15-20cm.

6. The method for storing Aspergillus brasiliensis spores according to any one of claims 1-5, characterized in that, The *Aspergillus brasiliensis* strain mentioned is the standard strain ATCC 16404.

7. The method for storing Aspergillus brasiliensis spores according to claim 1, characterized in that, The 1L nutrient solution contains the following components: 0.7g KH2PO4, 0.3g K2HPO4, 0.5g MgSO4·7H2O, 2g NaNO3, 0.01g FeSO4·7H2O, 0.5g KCl and 30g sucrose, with the remainder being water.

8. The method for storing Aspergillus brasiliensis spores according to claim 1 or 2, characterized in that, The cross-linked polyethylene material with dormant Aspergillus brasiliensis spores attached was sealed and stored at room temperature and pressure. The ambient temperature is 20–25℃ and the ambient pressure is 96.2–106.4 kPa.

9. The application of the Brazilian Aspergillus spore storage method according to any one of claims 1-8 in space station experiments.

10. A method for testing the degree of corrosion of cross-linked polyethylene materials by Aspergillus brasiliensis on a space station, characterized in that, This includes the method for storing Aspergillus brasiliensis spores as described in any one of claims 1-8; The Aspergillus brasiliensis spores obtained by the storage method of any one of claims 1-8 are activated by water injection, and the state of the cross-linked polyethylene material is observed.

Images