Application of yarrowia caribbica NJC36 in prevention and treatment of aspergillus flavus

Abstract

The invention discloses application of yarrowia caribbica NJC36 in prevention and treatment of aspergillus flavus, and belongs to the technical field of biological prevention and treatment. Experiments prove that the yarrowia caribbica NJC36 can inhibit the growth of aspergillus flavus and the synthesis of aflatoxin, and can be used for preventing and treating kiwi fruits from being infected by the aspergillus flavus. The yarrowia caribbica NJC36 disclosed by the invention has a remarkable prevention and treatment effect on aspergillus flavus, can inhibit the synthesis of aflatoxin, and is environment-friendly.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to the application of Calibick Mayer's yeast NJC36 in the control of Aspergillus flavus. Background Technology

[0002] Aspergillus flavus is widely distributed in the natural environment and can produce highly toxic aflatoxins (AFTs), with AFB1 being the most potent. AFB1 has proven carcinogenic, mutagenic, and teratogenic effects and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). These toxins widely contaminate various grain crops and foods, such as peanuts, millet, sesame, corn, wheat, rice, figs, spices, and cocoa, posing a serious threat to human and animal health. Due to its high thermal stability and wide distribution, AFTs easily accumulate during storage, transportation, and processing, making them difficult to remove. Reports of food or feed poisoning and high rates of liver cancer caused by Aflatoxin contamination are frequent, making it a global public health issue.

[0003] Aspergillus flavus not only possesses extremely strong environmental adaptability, but it can also rapidly multiply and produce a large number of spores in dry or humid and hot environments, making its control extremely difficult. The inventors first reported a case of Aspergillus flavus infecting kiwifruit in 2022 (Plant Disease, 2022, 106:1990). Field investigations showed that approximately 15% of mature kiwifruit were infected, indicating that the scope of this pathogen's damage is continuously expanding.

[0004] Current research indicates that yeasts have significant potential in controlling aflatoxin and its toxins. For example, literature reports that *Meyerozyma spp.* yeast can significantly inhibit *Aspergillus flavus* mycelial growth and spore formation, and reduce the production of aflatoxin AFB1 (Toxins-Basel, 2023, 15:402). Another study found that *Meyerozyma guilliermondii AF01* can not only adsorb AFB1 but also degrade the toxin (Biological Control, 2025, 200:105676), and effectively remove AFB1 from contaminated peanut meal in practical applications (Toxins-Basel, 2024, 16:305). Related studies have also identified specific genes required for this strain to degrade aflatoxin (such as aldo-keto reductase genes).

[0005] However, there are currently no literature reports on the application of *Meyerozyma caribbica* in the prevention and control of *Aspergillus flavus* or the degradation of aflatoxin. Therefore, this invention provides a new use for the *Meyerozyma caribbica* strain, which can not only significantly inhibit the growth of *Aspergillus flavus* mycelium and toxin production, but may also have a unique aflatoxin degradation mechanism, showing good application prospects and promotional value. Summary of the Invention

[0006] One of the objectives of this invention is to provide the application of Meyerozyma caribbica NJC36 in inhibiting the growth of Aspergillus flavus.

[0007] In this invention, the Meyerozyma caribbica NJC36 has the accession number CCTCC M 20221477, the accession date is September 23, 2022, and the depositary is the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Co-culturing NJC36 with Aspergillus flavus showed that NJC36 could compete with Aspergillus flavus for nutrients and growth space, thus achieving an antibacterial effect. Analysis of the culture supernatant of NJC36 revealed the presence of 2-phenylethanol, which inhibited the growth of Aspergillus flavus.

[0009] The second objective of this invention is to provide the application of Meyerozyma caribbica NJC36 in the postharvest control of Aspergillus flavus in kiwifruit.

[0010] In one embodiment of the present invention, the concentration is 1×10 7 Spraying a CFU / mL suspension of NJC36 bacteria onto kiwifruit can achieve a 91.4% therapeutic effect against Aspergillus flavus.

[0011] A third objective of this invention is to provide the application of Meyerozyma caribbica NJC36 in inhibiting aflatoxin synthesis and / or degrading aflatoxin.

[0012] Furthermore, the aflatoxin is aflatoxin B1.

[0013] This invention demonstrates through experiments that *Callibik Mayer's yeast* NJC36 can inhibit the growth of *Aspergillus flavus* and the synthesis of aflatoxin, and can be applied to prevent *Aspergillus flavus* infection in kiwifruit. The *Callibik Mayer's yeast* NJC36 of this invention has a significant effect on the prevention and control of *Aspergillus flavus*, can inhibit the synthesis of aflatoxin, and is environmentally friendly. Attached Figure Description

[0014] Figure 1 The results of trypan blue and neutral red staining of Aspergillus flavus hyphae during the co-culture of yeast NJC36 and Aspergillus flavus in Example 1 are shown.

[0015] Figure 2 The results show the identification of the active product synthesized from NJC36 in Example 2, where A is the HPLC chromatogram and B is the MS / MS identification chromatogram.

[0016] Figure 3 Example 3 illustrates the inhibitory effect of NJC36 on aflatoxin, where A represents the concentration analysis of AFB1 in the co-culture of NJC36 and Aspergillus flavus, and B represents the effect of qRT-PCR on the expression of aflatoxin synthesis-related genes in Aspergillus flavus.

[0017] Figure 4 Example 4 shows the detection of aflatoxin AFB1 degradation by NJC36. In the figure, A is the LC-MS detection of AFB1 degradation, B is the MS / MS analysis chromatogram of AFM1, the degradation product of AFB1 after NJC36 treatment, C is the MS / MS analysis chromatogram of AFP1, the degradation product of AFB1 after NJC36 treatment, D is the concentration of AFM1 after NJC36 treatment, and E is the concentration of AFP1 after NJC36 treatment.

[0018] Figure 5 This is the effect of NJC36 on the diameter of Aspergillus flavus lesions on kiwifruit in Example 5. A shows images of lesions treated with different concentrations of NJC36; B shows the statistical analysis of the effect of different concentrations of NJC36 on lesion length; C shows the qualitative analysis of AFB1 on kiwifruit treated with different concentrations of NJC36 using LC-MS; and D shows the quantitative analysis of AFB1 content on kiwifruit treated with different concentrations of NJC36 using LC-MS.

[0019] Figure 6 This is an analysis of the protective effect of NJC36 on zebrafish exposed to AFB1 in Example 6. In this example, A represents the malformations detected in zebrafish embryos after AFB1 treatment, B represents the survival rate of zebrafish on days 0, 1, 2, 3, and 4 after NJC36 treatment, C represents the heart rate of zebrafish embryos on days 0, 1, 2, 3, and 4 after NJC36 treatment, and D represents the body length of zebrafish 4 days after NJC36 treatment. Detailed Implementation

[0020] 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.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] The culture medium formulations used in the following examples are as follows:

[0024] 1. PDA solid culture medium (1L)

[0025] reagents quality potato 200g glucose 20g Agar powder 15g distilled water 1L

[0026] Prepare the ingredients according to the formula, boil for about 30 minutes, and after all components have dissolved, sterilize at 121°C for 15 minutes using the moist heat sterilization method.

[0027] 2. PDA liquid culture medium (1L)

[0028] reagents quality potato 200g glucose 20g distilled water 1L

[0029] Prepare the ingredients according to the formula, boil for about 30 minutes, and after all components have dissolved, sterilize at 121°C for 15 minutes using the moist heat sterilization method.

[0030] 3. YEPD medium

[0031] reagents quality Yeast extract 150mg trypsin 500mg glucose 1g distilled water 50mL

[0032] After each component is dissolved, it is sterilized at 121°C for 15 minutes using a moist heat sterilization method.

[0033] Example 1

[0034] Antagonistic effect of NJC36 against Aspergillus flavus

[0035] NJC36 was cultured in 50 mL of YEPD medium in a shaker at 28°C for 2 days to prepare OD. 600=1.5% cell suspension. Preparation of Aspergillus flavus mycelial cakes: The Aspergillus flavus strain NJC03 (Plant Disease, 2022, 106:1990) isolated in the inventor's laboratory was inoculated into PDA solid medium and cultured in a constant temperature incubator at 28℃ in the dark for 3 days. Mycelial cakes were then cut from the colony edges using a 3mm diameter punch to prepare mycelial cakes for later use. The Aspergillus flavus mycelial cakes were inoculated onto PDA solid medium, and 15μL of YEPD liquid medium containing NJC36 was spread in a straight line, with 3 replicates for each pathogenic fungus. The medium was incubated at 28℃ in the dark for 3 days. After 3 days, Aspergillus flavus hyphae from the proximal and distal ends of NJC36 were picked from the medium. The proximal and distal hyphae were stained with neutral red and trypan blue, respectively, and the staining was observed under a microscope.

[0036] Trypan blue staining: Wash the glass slide, add 1 μL of sterile water, take a small amount of hyphae and put them into the sterile water, absorb the excess water with filter paper, lightly flame the slide a few times to fix the sample, add 2-3 drops of trypan blue staining solution to cover the sample, stain for 8-10 minutes, then absorb the staining solution, wash 2-3 times with sterile water, cover with a coverslip, and observe under a microscope. Live cells should be colorless, while dead cells will be stained blue.

[0037] Neutral red staining: Take a clean glass slide, add 1 μL of sterile water, take a small amount of mycelium into the sterile water, absorb the excess water with filter paper, lightly flame the slide a few times to fix the sample, add 2-3 drops of neutral red staining solution to cover the sample, stain for 8-10 minutes, then absorb the staining solution, wash 2-3 times with sterile water, and observe under a microscope. Live cells will stain red, while dead cells will be colorless.

[0038] Observation results as follows Figure 1 As shown, Aspergillus hyphae near NJC36 stained dark blue with trypan blue but remained colorless with neutral red, indicating that the Aspergillus hyphae near NJC36 were dead. Aspergillus hyphae farther from NJC36 stained pale blue or colorless with trypan blue but red with neutral red, indicating that the Aspergillus hyphae farther from NJC36 were alive. The results indicate that NJC36 can kill adjacent Aspergillus flavus.

[0039] Example 2

[0040] Isolation and Identification of Active Products in NJC36

[0041] NJC36 was inoculated into YEPD medium and cultured on a shaker at 28°C and 200 rpm for 3 days. After centrifugation, the supernatant was collected and lyophilized. 0.1 g of the lyophilized powder was weighed, dissolved in 1 mL of methanol, filtered, and detected by HPLC and LC-MS (C18 reverse-phase column, mobile phase: 65:25:10 water / methanol / acetonitrile mixture, flow rate: 1 mL / min). The injection volume was 10 μL, and three replicates were set up. The results are as follows: Figure 2 As shown, the HPLC chromatogram shows a UV absorption peak at 16.6 min. Figure 2 (A) The substance at this peak was collected, evaporated to dryness, and then used for mass spectrometry detection. A 121.1 m / z peak was found in the anion mode of the first-stage full-scan mass spectrum. Second-stage mass spectrometry analysis of this ion revealed ion fragments at 91.1, 77, and 65.1 m / z. Based on ion fragment analysis, the product is likely 2-phenylethanol (…). Figure 2 (B) Liquid chromatography analysis of the standard revealed a peak elution time of 16.6 min, consistent with the peak elution time of the active substance in the lyophilized powder. Figure 2 (A) indicates that NJC36 can synthesize 2-phenylethanol.

[0042] Example 3

[0043] NJC36 can inhibit the synthesis of aflatoxin.

[0044] Existing research has shown that AFB1 is the most toxic aflatoxin and the only aflatoxin classified as a human carcinogen by the International Agency for Research on Cancer. AFG1, AFB2, and AFG2 are the next most toxic, with AFB1, AFG1, and AFB2 being the main toxins synthesized by Aspergillus flavus. Aflatoxin synthesis is regulated by multiple genes, including aflC, aflK, aflL, aflE, and aflO.

[0045] The preparation of Aspergillus flavus mycelial cakes is the same as in Example 1. Take 3 mycelial cakes and add them to a solution containing 10... 4 10 5 10 6 10 7 The cells were cultured in 50 mL YEPD medium containing CFU / mL NJC36 cells. A negative control was set up without NJC36 cells. The bacterial culture was incubated at 26°C with shaking at 200 rpm for 3 days. After centrifugation, the supernatant was collected, lyophilized, and the contents of AFB1, AFB2, and AFG2 were detected by LC-MS. The co-cultured cells were washed with sterile water, and total RNA was extracted. The expression of aflatoxin synthesis-related genes was detected by q-RT PCR. A negative control was set up without NJC36 cells.

[0046] The results showed that when the number of NJC36 cells was 10... 410 5 10 6 and 10 7 At CFU / mL, the concentration of AFB1 in the co-culture was significantly reduced. Figure 3 (A) In 10 6 and 10 7 In the presence of NJC36 cells with CFU / mL, the concentration of AFB1 was reduced by 53.8% and 61.4% compared with the negative control group, respectively.

[0047] In addition, NJC36 can continuously reduce the expression of multiple aflatoxin biosynthesis genes. Figure 3 (B) includes AflC (polyketide synthase), AflE (noraflatoxin acid reductase), AflK (chromotin synthase), AflL (desaturase), and AflO (O-methyltransferase). Using 10 7 Treatment of NJC36 cells with CFU / mL resulted in a 0.36-fold, 0.27-fold, 0.25-fold, 0.33-fold, and 0.11-fold decrease in the expression of AflC, AflE, AflK, AflL, and AflO, respectively. This indicates that NJC36 not only reduces the growth of Aspergillus flavus mycelium but also inhibits the biosynthesis of AFB1.

[0048] Example 4

[0049] NJC36 degrades AFB1 into AFM1 and AFP1.

[0050] It will contain 2.5 mg / mL AFB1 and 10 4 10 5 10 6 and 10 7 YEPD solution (50 mL) containing CFU / mL NJC36 cells was shaken at 200 rpm for 12 hours. A negative control was set up without NJC36 cells. After centrifugation, the culture was collected, and the AFB1 content was directly quantified by LC-MS. Degradation products AFM1 and AFP1 were identified by MS / MS and LC-MS. The detection conditions for AFM1 and AFP1 were the same as those for AFB1 quantification using liquid chromatography-mass spectrometry (LC-MS). AFM1 was quantified in positive ion mode using fragment ions from 329.1 to 229.0 ppm; AFP1 was quantified in positive ion mode using fragment ions from 299.0 to 215.0 ppm. The peaks corresponding to AFM1 and AFP1 were detected at 6.9 min and 7.7 min, respectively.

[0051] Without NJC36 treatment, the degradation rate of AFB1 was only 4.2% after 12 hours, but when the number of NJC36 cells was 10... 4 10 5 10 6and 10 7 At CFU / mL, the degradation rate of AFB1 increased to 34.2%, 51.0%, 61.2%, and 76.0%, respectively, indicating that NJC36 can not only inhibit the synthesis of AFB1 by Aspergillus flavus, but also degrade AFB1. Figure 4 (A). To identify AFB1 degradation products after NJC36 treatment, the potential presence of all previously reported AFB1 degradation products was further investigated. Results showed that AFM1 and AFP1 were present in the culture medium. Figure 4 (Medium B and 4 Medium C). However, no other degradation products were detected. AFM1 was detected in both the presence and absence of NJC36 treatment, indicating that AFB1 is degraded to AFM1 in the culture medium. Compared with the control group, 10 7 CFU / mL NJC336 cell treatment increased AFM1 concentration by 88.7% ( Figure 4 (D). Conversely, AFP1 was only detected in the NJC36 treatment, while it was not detected in the negative control group.

[0052] Analysis showed that the m / z of the main peak of AFM1 was 329.1, which is consistent with the calculated exact mass number (C). 17 H 13 O7[M+H]+=329.0656) is consistent ( Figure 4 (B) MS / MS analysis of the 329.1 peak revealed fragments at 313.1, 273.0, 258.1, and 229.1. On the other hand, AFP1 analysis yielded a main peak m / z of 299.1, consistent with the calculated AFP1 exact mass number (C). 16 H 11 O6[M+H]+=299.0556) is consistent ( Figure 4 (C). MS / MS analysis of the 299.1 peak revealed fragments at 281.1, 271.1, 215.1, and 170.1. After NJC36 treatment, the AFM1 concentration was 0.15–0.22 mg / L. Figure 4 The concentration of AFP1 in the medium (D) ranged from 0.72 to 1.67 mg / L. Figure 4 (E). This indicates that NJC36 primarily converts AFB1 to AFP1. Using 10 7After 12 h of treatment with NJC36 cells at CFU / mL, the concentrations of AFM1 and AFP1 were 0.22 ± 0.01 and 1.67 ± 0.19 mg / L, respectively (AFM1 concentration: 0.67 μM; AFP1 concentration: 5.60 μM). This is consistent with the degradation rate of AFB1 after 12 h (initial AFB1 concentration: 8.43 μM; AFB1 concentration after 12 h: 2.02 μM; AFB1 concentration after degradation by NJC36 cells: 6.41 μM), confirming that AFM1 and AFP1 are the main degradation products of AFB1 during NJC36 treatment.

[0053] Example 5

[0054] NJC36 can inhibit the growth of Aspergillus flavus on kiwifruit.

[0055] The method for preparing Aspergillus flavus cakes is the same as in Example 1. Three Aspergillus flavus cakes were inoculated into 50 mL of PDA liquid medium and cultured for 3 days at 28°C and 200 rpm in a shaker. The Aspergillus flavus solution was evenly sprayed onto the surface of kiwifruit (10 mL per kiwifruit). After air-drying, an equal volume of NJC36 cell suspension (10% concentration) was sprayed onto the kiwifruit. 4 10 5 10 6 10 7 The concentration of CFU / mL was measured, with sterile water spraying serving as a negative control. The treated kiwifruit were stored in the dark for 4 days, and the diameter of the lesions was measured and recorded.

[0056] The application of NJC36 significantly reduced the rot symptoms caused by Aspergillus flavus in kiwifruit. Figure 5 (A and B). Apply 10 6 and 10 7 After inoculation with NJC36 cells at CFU / mL, the lesion length was shortened by 47.5% and 91.4%, respectively. NJC36 also inhibited the accumulation of aflatoxin B1 (AFB1) in kiwifruit, reducing its content by 88.8% and 96.1%, respectively. Figure 5 (C and D). These results indicate that NJC36 can be used to control aflatoxin B1 in fruits.

[0057] Example 6

[0058] NJC36 can inhibit the toxicity of aflatoxin AFB1 in zebrafish.

[0059] Treatment of zebrafish with an aqueous solution containing AFB1 negatively impacts zebrafish embryonic development. The dosage of NJC36 was 10... 4 10 5 10 6 and 10 7CFU / mL. No NJC36 was added in the control experiment. After culturing at 26℃ and 200 rpm for 1 day, NJC36 cells were discarded by centrifugation at 8,000 g and 4℃ for 10 minutes. The supernatant was collected, sterilized using a 0.22 μm nylon filter (Jinteng, China), and diluted 1000-fold with ddH2O. The toxicity of the resulting solution was screened using a zebrafish model according to OECD and ISO 15088 standards. Zebrafish embryos were cultured in ddH2O at 25℃, pH 7.0, and conductivity 550 ± 50 μS for 4 hours. Then, the aqueous solution was replaced with the treatment solution. Zebrafish were exposed to the solution individually. Body length of the zebrafish was measured every 24 hours from day 2 to day 4. Heart rate and mortality were measured after day 4. Body length, heart rate, and mortality are commonly used parameters after AFB1 treatment in zebrafish. Photographs were taken using a Nikon SMZ445 stereomicroscope on days 2, 3, and 4 after incubation. Twenty zebrafish embryos were used at each treatment condition and time point. The experiment was repeated three times (n=60).

[0060] Two days after zebrafish hatching, treatment with AFB1 resulted in a shortening of the yolk sac and the elongated portion of the yolk sac in the zebrafish embryos. Figure 6 (A). However, in 10 4 10 5 10 6 and 10 7 No such effect was observed with CFU / mL NJC36 cell treatment. AFB1 treatment caused zebrafish to bend their body axis at 10 4 CFU / mL was detectable in NJC36 cells, but not in other treatments. After 4 days of culture in the presence of AFB1, the zebrafish embryo mortality rate was as high as 21.7%. Figure 6 (B) However, 10 4 10 5 10 6 and 10 7 Treatment with CFU / mL NJC36 cells reduced zebrafish mortality to 15.0%, 15.0%, 5.0%, and 0%, respectively. 7 The application of NJC36 cells at CFU / mL completely eliminated the lethal effect of AFB1 on zebrafish, indicating that NJC36 significantly reduced the toxicity of aflatoxin AFB1. On the other hand, 10 4 10 5 10 6 and 10 7 Treatment with CFU / mL NJC36 cells resulted in increases in zebrafish embryo heart rate of 22.0%, 27.4%, 27.8%, and 37.3% per minute, respectively. Figure 6(C). AFB1 toxicity also affects the body length of zebrafish embryos after days 3 and 4 of culture. On day 4 of culture, 10 4 10 5 10 6 and 10 7 Compared with the control group, the body length of zebrafish treated with CFU / mL NJC36 cells increased by 22.0%, 27.4%, 27.8%, and 37.3%, respectively. Figure 6 (D). Since heart rate and body length are important parameters related to zebrafish growth and development, this result indicates that NJC36 treatment can minimize the negative impact of AFB1-containing solutions on zebrafish development.

Claims

1. Calibick Mayer's yeast ( Meyerozyma caribbica Application of NJC36 in inhibiting the growth of Aspergillus flavus.

2. Calibick Mayer's yeast ( Meyerozyma caribbica Application of NJC36 in the postharvest control of Aspergillus flavus in kiwifruit.

3. The application according to claim 2, characterized in that, The application involves spraying a suspension of NJC36 bacteria onto the surface of kiwifruit to control Aspergillus flavus.

4. The application according to claim 3, characterized in that, The concentration of the NJC36 bacterial suspension was 1 × 10⁻⁶. 7 CFU / mL.

5. Calibick Mayer's yeast ( Meyerozyma caribbica Application of NJC36 in inhibiting aflatoxin synthesis and / or degrading aflatoxin.

6. The application according to claim 5, characterized in that, The aflatoxin in question is aflatoxin B1.

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