A method of enhancing the sensitivity of paecilomyces fungi to ultraviolet radiation

Abstract

The application discloses a method for enhancing the sensitivity of Paecilomyces fungi to ultraviolet radiation, and belongs to the technical field of biology. Paecilomyces Based on the discovery that the biosynthesis of the tough core component pyridine dicarboxylic acid (DPA) of Paecilomyces fungi spores depends on iron-mediated enzyme activity, free iron ions are effectively stored and DPA synthesis is inhibited by applying a specific concentration of iron chelator, so that the sensitivity of Paecilomyces fungi spores to ultraviolet (UV-A and UV-B) radiation is significantly enhanced. Paecilomyces The application provides a low-cost, efficient and environment-friendly fungal control strategy, and has important application value in the fields of food safety, agriculture and industrial sterilization.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an enhancement of the Paecilomyces genus (…). Paecilomyces Methods for assessing the sensitivity of fungi to ultraviolet radiation. Background Technology

[0002] Penicillium ( Paecilomyces Fungi are a widely distributed and highly representative group of food spoilage bacteria. Especially during the storage of fresh agricultural products and processed foods, the strong environmental resilience exhibited by their spores poses a serious challenge to food safety. Ultraviolet (UV) radiation, as a non-thermal physical sterilization technology, is widely used in the food industry due to its advantages such as high efficiency and no residue. However, *Paecilomyces* spores exhibit significant UV resistance; conventional UV irradiation doses are often insufficient to completely kill them, allowing residual spores to germinate under suitable conditions and cause food spoilage.

[0003] Current research indicates that the high resilience of *Paecilomyces* spores is closely related to the accumulation of pyridine dicarboxylic acid (DPA) within them. DPA is a key component of fungal spores' environmental stress resistance, effectively protecting them from various environmental stresses, including ultraviolet radiation. Previous research by the inventors has identified *Paecilomyces giantiflora* (… Paecilomyces maximus The core gene pmdpa, responsible for the synthesis of DPA, was identified and its important role in fungal colonization was confirmed.

[0004] Although genetic studies have revealed the importance of DPA synthesis, using genetic modification to control spoilage bacteria is not feasible in practical production applications. DPA synthase is an iron-dependent enzyme, and the utilization rate of free iron ions is the main limiting factor for DPA production. How to interfere with the biosynthesis of DPA in actual production processes using non-genetic methods, thereby weakening the inherent resistance of pathogens, is a pressing technical challenge that needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to overcome the limitations of existing technologies in the Penicillium genus (…). Paecilomyces To address the shortcomings of fungal spores, such as strong UV resistance and difficulty in complete removal by conventional disinfection methods, this paper proposes a method to enhance the sensitivity of this genus of fungi to UV radiation by applying an iron chelating agent.

[0006] To achieve the above objectives, the present invention employs the following technical means: A type of enhanced Paecilomyces ( Paecilomyces A method for assessing the sensitivity of fungi to ultraviolet radiation, which involves using iron chelating agents to target Paecilomyces ( ). Paecilomyces The fungi are treated to enhance their sensitivity to ultraviolet radiation.

[0007] Furthermore, the iron chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), citric acid, picolinic acid, and 1,10-phenanthroline.

[0008] Furthermore, the *Penicillium* genus ( Paecilomyces The fungus is *Paecilomyces megaterium* (…). Paecilomyces maximus NJC01. This giant Paecilomyces cerevisiae is deposited at the China Center for Type Culture Collection (CCTCCM) with accession number CCTCM 2026594, deposited on April 3, 2026, at Wuhan University, Wuhan, China.

[0009] Furthermore, the iron chelating agent inhibits the growth of *Paecilomyces* species by encapsulating free iron ions in the environment. Paecilomyces The biosynthesis of endogenous pyridine dicarboxylic acid (DPA) in fungi weakens the environmental resistance of fungal spores.

[0010] Further, the treatment involves removing the Paecilomyces genus ( Paecilomyces The fungi were pretreated by inoculating them in a medium containing an iron chelating agent at a concentration of 0.05-5 mmol / L.

[0011] Furthermore, the concentration of the iron chelating agent is selected as follows: When the chelating agent is ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid, its effective concentration is 1-5 mmol / L; When the chelating agent is citric acid or pyridinecarboxylic acid, its effective concentration is 0.05 - 0.5 mmol / L; When the chelating agent is 1,10-phenanthroline, its effective concentration is 0.05 mmol / L.

[0012] Furthermore, the ultraviolet radiation is selected from UV-A, UV-B, or a combination of both.

[0013] Furthermore, the peak wavelength of UV-A is 340 nm, and the irradiation time is 24 h; the peak wavelength of UV-B is 313 nm, and the irradiation time is 48 h.

[0014] Iron chelating agents in the preparation of agents for enhancing Paecilomyces ( PaecilomycesApplication in compositions for fungal UV-sensitive organisms, wherein the iron chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), citric acid, picolinic acid, and 1,10-phenanthroline.

[0015] The core of this invention lies in interfering with the biosynthesis of pyridine dicarboxylic acid (DPA), a key component of spore toughness, through iron chelation, thereby weakening its environmental resistance.

[0016] The inventors determined through experiments that it could effectively inhibit Paecilomyces megaterium (… Paecilomyces maximus The types of iron chelating agents synthesized from DPA in NJC01 were investigated to reduce its UV resistance. The optimal application concentration of the iron chelating agents was further determined to maximize the inhibitory effect on fungal growth and the UV sensitizing effect. By disclosing the synergistic killing effect between iron chelation treatment and UV exposure (UV-A and UV-B), a feasible solution for agricultural and industrial pest control is provided.

[0017] The iron chelating agent is selected from one or more of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), citric acid, picolinic acid, and 1,10-phenanthroline. This chelating agent is used to immobilize free iron ions in the fungal environment, thereby inhibiting iron-dependent DPA synthase activity. Since DPA is a key substance for maintaining fungal spore resistance, iron deficiency leads to inhibited DPA biosynthesis, thus making the fungus sensitive to environmental stress.

[0018] The specific processing technology is as follows: Pretreatment: *Paecilomyces* fungi (e.g., strain NJC01) were inoculated into a culture medium containing an effective concentration of iron chelating agent. Specifically, the concentration of 1,10-phenanthroline was 0.05 mmol / L; the concentrations of pyridinecarboxylic acid and citric acid were 0.05-0.5 mmol / L; and the concentrations of EDTA and DTPA were 1-5 mmol / L.

[0019] Ultraviolet (UV) exposure: The fungal spores treated with the chelating agent were irradiated with UV light. The UV light included UV-A with a peak wavelength of 340 nm (irradiation for 24 h) and / or UV-B with a peak wavelength of 313 nm (irradiation for 48 h).

[0020] This invention, by introducing specific iron chelating agents (such as EDTA, DTPA, and 1,10-phenanthroline), cleverly utilizes an iron deprivation strategy to inhibit DPA synthesis, thereby significantly enhancing the sensitivity of *Paecilomyces* fungi to ultraviolet radiation. This synergistic strategy of chemical intervention and physical sterilization not only provides a novel and feasible method for the effective control and eradication of *Paecilomyces* in agricultural and industrial applications, but also provides a theoretical basis for the development of environmentally friendly new sterilization synergists.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. Significant synergistic effect: This invention reveals for the first time the synergistic effect of iron deprivation strategy and ultraviolet exposure. Experiments show that ultraviolet irradiation alone is insufficient to completely kill Paecilomyces spores, but after pretreatment with an iron chelating agent, the spore survival rate shows a dose-dependent and dramatic decrease under both UV-A and UV-B exposure.

[0022] 2. High efficiency and specificity: In particular, 1,10-phenanthroline exhibits excellent inhibitory activity even at extremely low concentrations (0.05 mmol / L). Furthermore, EDTA and DTPA, in combination with UV-B irradiation at a concentration of 5 mmol / L, produce the most significant growth-inhibiting effect.

[0023] 3. Non-hereditary industrial feasibility: The method of this invention does not require complex genetic modification of pathogens. The resistance of pathogens can be weakened through biochemical pathways by directly applying chemical chelating agents. The process is simple and low-cost.

[0024] 4. Biosafety: Some of the chelating agents (such as citric acid) used in this invention are environmentally friendly substances, providing a new green strategy for precise sterilization in food processing and agricultural product preservation. Attached Figure Description

[0025] Figure 1 The effects of various iron chelating agents on the growth of Paecilomyces NJC01 are shown. A and B show the results of treatment with EDTA and DTPA, respectively, while C, D, and E show the results of treatment with citric acid, pyridinecarboxylic acid, and 1,10-phenanthroline, respectively.

[0026] Figure 2 The effect of UV irradiation following EDTA treatment on Paecilomyces NJC01. A and B show the morphological results after UV-B and UV-A exposure, respectively, and C and D represent the colony count for each treatment group.

[0027] Figure 3 The effect of UV irradiation following DTPA treatment on Paecilomyces NJC01 is shown. A and B represent the morphological results after UV-B and UV-A exposure, respectively, while C and D represent the colony count for each treatment group.

[0028] Figure 4 The effect of citric acid treatment followed by ultraviolet irradiation on Paecilomyces NJC01 is shown. A and B show the morphological results after UV-B and UV-A exposure, respectively, and C and D represent the colony count for each treatment group.

[0029] Figure 5 The effect of UV irradiation on Paecilomyces NJC01 after pyridine carboxylic acid treatment is shown. A and B show the morphological results after UV-B and UV-A exposure, respectively, and C and D represent the colony count for each treatment group.

[0030] Figure 6 The effects of UV-B (A and C) and UV-A (B and D) radiation on *Paecilomyces cerevisiae* NJC01 treated with 1,10-phenanthroline are shown, illustrating colony growth morphology and corresponding colony numbers. Because 1,10-phenanthroline exhibits potent antifungal activity at high concentrations, samples treated with 0.05 mmol / L, 0.1 mmol / L, 0.2 mmol / L, and 0.5 mmol / L concentrations showed no growth under the culture conditions shown and are therefore not included in the figures. The data shown in the figures represent comparative results obtained at lower concentrations or under different conditions. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0034] Iron chelation therapy This embodiment uses PDA as a reference for Paecilomyces megaterium (… Paecilomyces maximus The basal culture medium for strain NJC01 was prepared as follows: 200 g of sliced ​​potatoes were soaked in boiling distilled water for 30 min, and the mixture was filtered through gauze to collect the aqueous extract. Subsequently, 20 g of glucose was added to the extract, and distilled water was added to bring the total volume to 1.0 L.

[0035] Using agarose as a solidifying agent, 1.5 g of agarose powder was weighed and added to 10 individual containers containing 100 mL of PDB, resulting in 10 100 mL units. All containers were autoclaved. To prepare plates, 10 mL of melted PDB medium was poured into sterile petri dishes. If a chelating agent was to be added, it was added to the liquefied medium and mixed thoroughly before dispensing to ensure even distribution in the medium.

[0036] The chelating agents used and their concentrations are as follows:

[0037] Experimental plates were prepared using 10 mL of PDA as a base, with different concentrations of chelating agents added. To avoid complete inhibition of strain NJC01 by high concentrations of chelating agents, the following concentration gradients were established for each chelating agent: pyridinecarboxylic acid and citric acid were added at 0.05, 0.1, 0.2, and 0.5 mmol / L, respectively; 1,10-phenanthroline, due to its strong inhibitory effect, was added at only 0.05 mmol / L; while EDTA and DTPA, with weaker inhibitory effects, were added at three higher concentrations of 1, 2, and 5 mmol / L. After inoculation, the cultures were incubated at 28°C for 72 h. The incubation process was carried out under complete darkness to optimize fungal development. After 72 h, the plates were removed for digital imaging and quantitative analysis.

[0038] like Figure 1 As shown, compared with the untreated control group, the mycelial proliferation in the treated group showed a significant dose-dependent decrease, and the morphology also underwent significant changes. Ethylenediaminetetraacetic acid (EDTA) Figure 1 A) Diethylenetriaminepentaacetic acid (DTPA) Figure 1 Treatment with EDTA at concentrations of 1 mmol / L, 2 mmol / L, and 5 mmol / L significantly inhibited fungal growth. Specifically, EDTA at concentrations of 1, 2, and 5 mmol / L reduced fungal growth by 35%, 60%, and 75%, respectively. Valeric acid treatment reduced fungal growth by 60% across the tested concentration range (1–5 mmol / L). Notably, valeric acid resulted in a concentration-dependent decrease in yellow pigment, indicating interference with the biosynthesis of secondary metabolites. Citric Acid (…) Figure 1 The inhibitory activity of C) is negligible; pyridinecarboxylic acid (Picolinic Acid) Figure 1 The middle D) showed a significant inhibitory effect, with a growth inhibition rate of up to 56% observed in the concentration range of 0.05 mmol / L to 0.5 mmol / L. 1,10-Phenanthroline ( Figure 1Phenanthroline (E) exhibited the strongest inhibitory activity. At a concentration of 0.05 mmol / L, phenanthroline caused a 73% growth inhibition; when the concentration was further increased to 0.1 mmol / L and 0.5 mmol / L, the compound completely inhibited biosynthesis, thereby causing the fungus to completely stop proliferating. Example 2

[0039] Study on the irradiation of Paecilomyces fungi treated with chelating agents under UVA and UVB. Spores of *Paecilomyces* strain NJC01, treated with various chelating agents as described in Example 1, were harvested using a 0.1% (v / v) Tween® 20 aqueous solution. The standard concentration of the resulting spore suspension was adjusted to 1.0 × 10⁻⁶. 6 The spore count / mL was calculated and transferred to 1 mL microcentrifuge tubes for subsequent irradiation. The samples were then placed in a dedicated dark chamber and irradiated with UV-A light (peak wavelength 340 nm) for 24 h at a distance of 5 cm from the UV light source, and with UV-B light (peak wavelength 313 nm) for 48 h. A control group (CTL) was established under the same conditions without UV irradiation.

[0040] Following the set irradiation intervals, the suspension was inoculated onto PDA medium without additional chelating agents and cultured. The culture plates were incubated at 28°C for 48 h to promote fungal proliferation. Subsequent morphological development was continuously observed, and colonies were counted using the plate count method to analyze survival rates.

[0041] like Figure 2 As shown in Figures A and B, the survival rate of *Paecilomyces* treated with EDTA did not significantly decrease after 24 h of UV-A or UV-B irradiation; however, after 48 h of irradiation, the colony-forming units (CFU) decreased significantly in a dose-dependent manner. After 48 h of UV-B irradiation, at concentrations of 1 mmol / L, 2 mmol / L, and 5 mmol / L, fungal colony formation decreased by 62%, 90%, and 100%, respectively. Figure 2 (C). Correspondingly, after 48 h of UV-A irradiation, at concentrations of 1 mmol / L, 2 mmol / L, and 5 mmol / L, spore viability decreased by 36%, 40%, and 80%, respectively. Figure 2 (D).

[0042] like Figure 3As shown in Figures A and B, the spores harvested from valeric acid-treated cultures showed a significant decrease in viability after 48 h of UV irradiation. Significant inhibitory effects were observed at all concentrations after 48 h of UV-B irradiation, with spore viability decreasing by 92%, 90%, and 94% at concentrations of 1 mmol / L, 2 mmol / L, and 5 mmol / L, respectively. Figure 3 (C). After 48 h of UV-A irradiation, although the colony-forming units (CFU) decreased, the effect was not as significant as that of UV-B, with reductions of 30%, 32%, and 29% at concentrations of 1, 2, and 5 mmol / L, respectively. Figure 3 (D).

[0043] Citric acid exhibited a significant sensitizing effect on ultraviolet radiation. 24-hour irradiation showed almost no change, while 48-hour irradiation led to a significant decrease in spore viability. After 48 hours of UV-B irradiation, near-complete inhibition (approximately 99% reduction) was achieved at all tested concentrations (0.05, 0.1, 0.2, and 0.5 mmol / L). Figure 4 (A and C). After 48 h of UV-A irradiation, a concentration-dependent decrease in spore number was observed, with decreases of 35% (0.05 mmol / L), 40% (0.1 mmol / L), 55% (0.2 mmol / L), and 71% (0.5 mmol / L), respectively. Figure 4 (B and D in the middle).

[0044] Compared to other chelating agents, pyridine carboxylic acid exhibited relatively low spore sensitization activity, with significant effects observed only at the highest tested concentrations. After 48 h of UV-B irradiation, pyridine carboxylic acid at concentrations between 0.05 and 0.2 mmol / L reduced colony-forming units (CFU) by approximately 20%, while treatment at 0.5 mmol / L resulted in a 67% reduction in CFU. Figure 5 (Figures A and C). After 48 h of UV-A irradiation, a significant reduction in colony count was observed only at a concentration of 0.5 mmol / L, where the colony count decreased by 95% after 48 h of irradiation (Figures B and D).

[0045] Because phenanthroxaline is highly toxic to mycelial growth, the effect on spore sensitivity was only evaluated at a concentration of 0.05 mmol / L (Figures 6A and 6B). At this concentration, after 48 h of co-treatment with UV-B and UV-A, spore viability was reduced by 80% compared to the untreated control group (Figures 6C and 6D).

Claims

1. A species that enhances the Penicillium genus ( Paecilomyces A method for assessing the sensitivity of fungi to ultraviolet radiation, characterized in that, The method involves using an iron chelating agent to target Paecilomyces ( ). Paecilomyces The fungi are treated to enhance their sensitivity to ultraviolet radiation.

2. The method according to claim 1, characterized in that, The iron chelating agent is selected from ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, citric acid, pyridinecarboxylic acid, and 1,10-phenanthroline.

3. The method according to claim 1, characterized in that, The genus *Penicillium* ( Paecilomyces The fungus is *Paecilomyces megaterium* (…). Paecilomyces maximus NJC01, accession number CCTCC M 2026594, accession date April 3, 2026.

4. The method according to claim 1, characterized in that, The iron chelating agent inhibits the growth of Paecilomyces by sealing free iron ions in the environment. Paecilomyces The biosynthesis of endogenous pyridine dicarboxylic acid in fungi is weakened, thereby impairing the biosynthesis of Paecilomyces ( ). Paecilomyces The environmental resistance of fungal spores.

5. The method according to claim 1, characterized in that, The treatment involves removing the Paecilomyces genus ( Paecilomyces The fungi were pretreated by inoculating them in a medium containing an iron chelating agent at a concentration of 0.05-5 mmol / L.

6. The method according to claim 5, characterized in that, The concentration of the iron chelating agent is selected as follows: When the chelating agent is ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid, its effective concentration is 1-5 mmol / L; When the chelating agent is citric acid or pyridinecarboxylic acid, its effective concentration is 0.05 - 0.5 mmol / L; When the chelating agent is 1,10-phenanthroline, its effective concentration is 0.05 mmol / L.

7. The method according to claim 1, characterized in that, The ultraviolet radiation is selected from UV-A, UV-B, or a combination of both.

8. The method according to claim 1, characterized in that, The peak wavelength of UV-A is 340 nm, and the irradiation time is 24 h; the peak wavelength of UV-B is 313 nm, and the irradiation time is 48 h.

9. Iron chelating agents in the preparation of agents to enhance the properties of Paecilomyces ( ). Paecilomyces Application in compositions that are sensitive to ultraviolet light in fungi.

10. The application according to claim 9, characterized in that, The iron chelating agent is selected from ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, citric acid, pyridinecarboxylic acid, and 1,10-phenanthroline.

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