Hanna yeast strain zafu hh1 and application thereof

Abstract

The application discloses a strain of Hanna yeast ZAFU HH1, which is classified and named as Hannaella zeae , and is preserved in the China General Microbiological Culture Collection Center on June 28, 2023, with a preservation number of CGMCC No. 27725. The application also discloses a bacterial agent prepared from the strain. The application further discloses applications of the strain and the bacterial agent prepared therefrom in converting zearalenone. The Hanna yeast ZAFU HH1 has excellent ZEN conversion capacity, and the conversion product is identified as a derivative with lower toxicity. The safety of the ZEN conversion product is evaluated by using Caenorhabditis elegans as a model organism, and it is proved that the conversion product has good biological safety. In addition, under low-temperature stress conditions and acid stress conditions, the Hanna yeast ZAFU HH1 still maintains a high ZEN conversion rate.

Description

Technical Field

[0001] This invention relates to the field of microbial transformation technology, specifically to a strain of Hannah yeast ZAFU HH1 and its applications. Background Technology

[0002] Zearalenone (ZEN), produced by Fusarium spp., is a potent estrogenic fungal toxin with carcinogenic, reproductive toxicity, and endocrine-disrupting effects, seriously threatening agricultural product safety and human and animal health. Current ZEN detoxification technologies include chemical methods which carry the risk of drug residues, physical methods which easily reduce the nutritional value of feed, and mainstream microbial methods which face bottlenecks such as low detoxification efficiency, unclear safety of conversion products, and poor stress tolerance. While yeast is considered an ideal degradation carrier due to its genetic stability, strong environmental adaptability, and lack of harmful metabolite production, existing yeast strains generally suffer from insufficient activity under stress, unsystematized transformation pathways, and unassessed product toxicity.

[0003] The microbial resources nurtured by the extreme environment of the Qinghai-Tibet Plateau (low temperature, strong ultraviolet radiation, low oxygen) have unique stress adaptation mechanisms, providing potential germplasm resources for developing novel ZEN transforming strains that combine high transformation capacity and environmental adaptability. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Hannah yeast ZAFU HH1 and its applications to address the shortcomings of existing technologies.

[0005] The present invention adopts the following technical solution: The first aspect of this invention provides a strain of Hannahs yeast ZAFU HH1, which is classified and named as follows: Hannaella zeae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 28, 2023, with accession number CGMCC No. 27725.

[0006] The second aspect of the present invention provides a bacterial agent prepared from the above-mentioned Hannah yeast ZAFU HH1.

[0007] Further, the bacterial agent is prepared by the following method: Hannah yeast ZAFU HH1 is cultured in liquid to obtain a Hannah yeast ZAFU HH1 culture broth; the Hannah yeast ZAFU HH1 culture broth is centrifuged, and the bacterial cells are collected; the bacterial cells are washed with sterile water and then resuspended in sterile water to obtain the bacterial agent, the concentration of which is 1×10⁻⁶. 6 cells / mL -1×10 9 cells / mL.

[0008] Furthermore, the bacterial agent is prepared by the following method: Hannah yeast ZAFU HH1 is inoculated into a liquid culture medium, including NYDB medium, and cultured at 28-30℃ and 150-200 rpm for 1-3 days to obtain a Hannah yeast ZAFU HH1 culture broth. The Hannah yeast ZAFU HH1 culture broth is centrifuged, and the bacterial cells are collected. The bacterial cells are washed with sterile water and then resuspended in sterile water to obtain the bacterial agent, the concentration of which is 1×10⁻⁶. 6 cells / mL -1×10 9 cells / mL.

[0009] The third aspect of this invention provides the application of the above-mentioned Hannah yeast ZAFU HH1 in the conversion of zearalenone.

[0010] The fourth aspect of this invention provides the application of the above-mentioned microbial agent in the conversion of zearalenone.

[0011] The fifth aspect of this invention provides the application of the above-mentioned Hannah yeast ZAFU HH1 in inhibiting Fusarium.

[0012] Furthermore, the Fusarium includes Fusarium graminearum.

[0013] The sixth aspect of the present invention provides the application of the above-mentioned microbial agent in inhibiting Fusarium.

[0014] Furthermore, the Fusarium includes Fusarium graminearum.

[0015] The beneficial effects of this invention are: This invention screened a strain of *Hannaconia hannaensis* ZAFU HH1 from soil in Tibet, which exhibited excellent ZEN conversion ability, achieving a conversion rate of 85.87% for ZEN at a concentration of 5 μg / mL after 36 hours. The conversion product was identified as a derivative with low toxicity: ZEN-14-β-D-glucopyranoside (C... 24 H 32 O 10 ) and ZEN-16-β-D-glucopyranoside (C 24 H 32 O 10The safety of the ZEN transformation product was evaluated using *C. elegans* as a model organism, confirming its good biocompatibility. Furthermore, under low-temperature and acidic stress conditions, *Hannaconia hannaensis* ZAFU HH1 maintained high ZEN conversion rates of 57.48% and 47.10%, respectively. This study indicates that the ZEN transformation mechanism of *Hannaconia hannaensis* ZAFU HH1 mainly involves the synergistic effect of intracellular transformation and extracellular metabolism. This invention provides a highly efficient strain resource and a novel transformation pathway for ZEN biotransformation. The *Hannaconia hannaensis* ZAFU HH1 of this invention has significant application potential in pharmaceuticals, food, and feed toxicity reduction, providing an alternative strategy and potential strain resource for developing novel biotransformation technologies. Attached Figure Description

[0016] Figure 1 The conversion ability of 11 yeast strains to ZEN was evaluated.

[0017] Figure 2 Colony morphology of Hannahs yeast ZAFU HH1 cultured on NYDA medium (A), scanning electron micrograph (B), phylogenetic tree (C).

[0018] Figure 3 The effects of ZAFU HH1 concentration on ZEN conversion by ZAFU HH1 in Hannah yeast (A) and the effects of ZEN concentration on ZEN conversion by ZAFU HH1 in Hannah yeast (B).

[0019] Figure 4 This image shows the in vitro inhibitory effect of Hannahs yeast ZAFU HH1 on wild-type Fusarium graminearum strain PH-1. A shows the colony morphology of wild-type Fusarium graminearum strain PH-1 on PDA medium, and B shows the colony diameter statistics of wild-type Fusarium graminearum strain PH-1 after 3 to 7 days of growth on PDA medium.

[0020] Figure 5 The effect of actinomycete ketone on the transformation of ZEN by Hannah yeast ZAFU HH1.

[0021] Figure 6 The effect of different treatment solutions on ZEN conversion of Hannah yeast ZAFU HH1.

[0022] Figure 7 These are the results of first-order spectral analysis of ZEN. Relative Abundance represents relative abundance.

[0023] Figure 8 These are the results of primary spectrum analysis for compound 1. Relative Abundance represents the relative abundance.

[0024] Figure 9These are the results of primary spectral analysis of compound 2. Relative Abundance represents relative abundance.

[0025] Figure 10 This refers to the metabolic pathway of the transformation products.

[0026] Figure 11 Safety evaluation of the product of ZEN transformation by Hannah's yeast ZAFU HH1. A shows the effect of the product of ZEN transformation by Hannah's yeast ZAFU HH1 on the number of Caenorhabditis elegans progeny; B shows the effect of the product of ZEN transformation by Hannah's yeast ZAFU HH1 on the body length of Caenorhabditis elegans; C shows a statistical graph of the body length of Caenorhabditis elegans at each stromal stage.

[0027] Figure 12 The effects of acid stress (A), temperature stress (B), acid stress on ZAFU HH1 transformation of ZEN by Hannah yeast (C), and temperature stress on ZAFU HH1 transformation of ZEN by Hannah yeast (D) were investigated.

[0028] Information on the preservation of biological materials

[0029] strain ZAFU HH1, classified and named Hannaella zeae It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is June 28, 2023, and the accession number is CGMCC No. 27725. Detailed Implementation

[0030] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] The culture medium formulations involved in the following examples are as follows: Selective culture medium for Bengal red fungi (1 L system): 31.6 g of Bengal red agar, add 1 L of distilled water, stir and heat to boiling until completely dissolved, then autoclave at 121℃ for 20 minutes.

[0032] NYDA medium (1 L system): 8 g / L beef extract, 5 g / L yeast powder, 10 g / L glucose, 20 g / L agar powder, diluted to 1 L with distilled water, sterilized at 121℃ for 20 minutes.

[0033] NYDB medium (1 L system): 8 g / L beef extract, 5 g / L yeast powder, 10 g / L glucose, diluted to 1 L with distilled water, sterilized at 121℃ for 20 minutes.

[0034] PDA medium (1 L system): Potato extract powder 6 g / L, glucose 20 g / L, chloramphenicol 0.1 g / L, agar 20 g / L, diluted with distilled water to 1 L, sterilized at 121℃ for 20 minutes.

[0035] The nematode growth medium, namely NGM medium (1L system): 3 g sodium chloride, 2.5 g peptone, 25 mL 1M potassium phosphate buffer, 20 g agar powder, and dilute to 1 L with distilled water. Sterilize at 121℃ for 20 minutes. After cooling to 40-50℃, add 1 mL cholesterol (concentration of 5 mg / mL, dissolved in ethanol), and add 0.4 mL each of 1M CaCl2 and 1M MgSO4.

[0036] 1M potassium phosphate buffer (1 L system): Weigh 108.3 g of KH2PO4 and 35.6 g of K2HPO4, bring the volume to 1 L with distilled water, and sterilize at 121℃ for 20 minutes.

[0037] M9 buffer solution (1 L system): 8 g Na2HPO4, 3 g KH2PO4, 5 g sodium chloride, 1 mL 1M MgSO4, diluted to 1 L with distilled water, sterilized at 121℃ for 20 minutes.

[0038] Unless otherwise specified, the petri dishes (plates) involved in the following examples are 90 mm in diameter.

[0039] Example 1: Isolation and Screening of Functional Strains 1. A total of 95 soil samples were collected from farmland / pastures in Amdo County, Baqing County, and Nyima County of Nagqu City, Medog County and Bomi County of Nyingchi City, and Gonggar County, Sangri County, and Qusong County of Shannan City, Tibet Autonomous Region, China. These samples were stored at 4℃ for later use. 1 g of each soil sample was weighed and dissolved in 100 mL of sterile water, and then serially diluted to obtain 10⁻⁶ dilutions. 2 10 3 and 10 4 A soil solution of times the concentration of 1000 ppm.

[0040] 2. Take 100 μL from each soil solution and spread it onto a plate containing selective medium for Bengal red fungus, and incubate at 28°C for 3-5 days.

[0041] 3. Select single colonies with different culture characteristics using an inoculation loop, perform streak purification on agar plates, and after confirming them as yeast by microscopic examination, inoculate them into NYDA medium slant for culture and preservation.

[0042] 4. Inoculate the yeast preserved on the slant into a 250 mL Erlenmeyer flask containing 50 mL of NYDB medium and incubate at 28℃ and 200 rpm for 1 day to obtain the yeast culture medium.

[0043] 5. After centrifuging the yeast culture at 4℃ and 5000 g for 10 minutes, collect the cells. Wash the cells three times with sterile water, then resuspend the cells in sterile water and adjust the concentration of the yeast suspension to 1×10⁻⁶. 8 ce1ls / mL (hemocytometer count), ready for use.

[0044] 6. Screening of Zearalenone (ZEN) Detoxified Strains (1) Add 1 mL of 1×10⁻⁶ nitric acid to a 250 mL Erlenmeyer flask containing 50 mL of NYDB medium. 8 Yeast suspension with cells / mL (the control group was treated with an equal volume of sterile water) was then added with an appropriate amount of ZEN standard solution (10 mg of ZEN standard powder was dissolved in 10 mL of methanol to a concentration of 1000 μg / mL; the same applies below), and the initial ZEN concentration was controlled at 5 μg / mL. The mixture was cultured at 28℃ and 150 rpm, and samples were taken at 0 hours and 48 hours of culture.

[0045] (2) Three replicates were set up for each yeast strain. Take 500 μL of sample, add 500 μL of anhydrous ethanol, mix well, and centrifuge at 4℃ and 5000 g for 10 minutes. Take the supernatant and filter it through a 0.22 μm filter membrane. The residual concentration of ZEN in the filtrate was determined by high performance liquid chromatography. The ZEN conversion rate was calculated by the ZEN conversion rate calculation formula, and yeast strains with high ZEN conversion rate were screened.

[0046] The formula for calculating ZEN conversion rate is:

[0047] In the formula, φ ZEN conversion rate; S 0 represents the peak area of ​​the chromatogram of each group after 0 hours of ZEN conversion; S n The peak areas are the peak areas of the chromatograms of each group after 48 hours of ZEN conversion.

[0048] (3) During the screening process, the transformation ability of different yeast strains to ZEN after 48 hours was as follows: Figure 1 As shown. Figure 1The transformation efficiency of 11 yeast strains for ZEN was demonstrated. Yeast strains numbered 7, 139, 195, and 219 showed low ZEN transformation rates, all below 20%; strains numbered 223, 224, 3, and 576 showed ZEN transformation rates below 50%; while strains numbered 147, 143, and 221 showed high ZEN transformation rates, all exceeding 60%. Yeast strain number 221 (i.e., strain ZAFU HH1, initially numbered 221 during screening and named ZAFU HH1 upon submission for bio-preservation) exhibited the highest ZEN transformation capacity with a transformation rate of 86.17%, and was therefore selected for further research.

[0049] Example 2 Identification of strain ZAFU HH1 1. Morphological characteristics of strain ZAFU HH1 When the diluted culture medium of strain ZAFU HH1 was spread onto NYDA medium and incubated at 28°C for 5 days, the colonies were round, slightly raised, milky white, matte, opaque, with neat edges and a smooth surface. Figure 2 A); Under scanning electron microscopy, ZAFU HH1 cells appear rod-shaped ( Figure 2 B).

[0050] 2. Molecular identification The 26S rDNA gene D1 / D2 fragment of strain ZAFU HH1 was sequenced, and the obtained gene sequence was analyzed by BLAST search in the NCBI database, and a phylogenetic tree was constructed. Figure 2 C). The results showed that strain ZAFU HH1 and Hannaella zeae YM26007 has the highest homology, reaching 99.66%.

[0051] Based on the colony morphology of strain ZAFU HH1 and the phylogenetic tree constructed using 26S rDNA, strain ZAFU HH1 was identified as *Hannaconia hannaensis*. Hannaella zeae It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27725.

[0052] Example 3: Conversion effect of Hannah yeast ZAFU HH1 on ZEN 1. Effect of ZAFU HH1 concentration on ZEN conversion by Hannah yeast ZAFU HH1 Hannah's yeast ZAFU HH1 was inoculated into NYDB medium and cultured at 28℃ and 200 rpm for 1 day to obtain Hannah's yeast ZAFU HH1 culture broth. The Hannah's yeast ZAFU HH1 culture broth was centrifuged at 4℃ and 5000 g for 10 minutes to collect the cells. The cells were washed three times with sterile water and then resuspended in sterile water. The concentration of the Hannah's yeast ZAFU HH1 suspension was adjusted to 1×10⁻⁶. 6 1×10 7 1×10 8 and 1×10 9 ce11s / m1 (hemocytometer counting), ready for use. Add 1 mL of adjusted Hannah's yeast ZAFU HH1 suspension (equal volume of sterile water for the control group) to 50 mL NYDB medium, then add an appropriate amount of ZEN standard solution to adjust the initial ZEN concentration to 5 μg / mL. Incubate at 28℃ and 150 rpm. Samples are collected at 0, 24, 36, 48, 60, and 72 hours. The residual ZEN concentration is determined using high-performance liquid chromatography (HPLC), and the ZEN conversion rate is calculated using the ZEN conversion rate calculation formula. Each group has three replicates.

[0053] The formula for calculating ZEN conversion rate is:

[0054] In the formula, φ ZEN conversion rate; S 0 represents the peak area of ​​the chromatogram of each group after 0 hours of ZEN conversion; S n The peak areas of the chromatograms of ZEN converted for n hours for each group (n=24, 36, 48, 60, 72).

[0055] like Figure 3 As shown in Figure A, Hannah's yeast ZAFU HH1 at the lowest concentration (1×10⁻⁶) 6 (cells / mL) and low concentration (1×10) 7 At a concentration of (1 × 10⁻⁶ cells / mL), ZEN conversion reached its peak at 48 hours. Hannah yeast ZAFU HH1 at a medium concentration (1 × 10⁻⁶ cells / mL) was also utilized. 8 An accelerated ZEN conversion rate was observed at a concentration of (1 × 10⁻⁶ cells / mL), peaking at 36 hours. Hannah yeast ZAFU HH1 at a high concentration (1 × 10⁻⁶ cells / mL) was used. 9At a concentration of cells / mL, ZEN conversion reached its peak within 24 hours. In microbial detoxification systems, excessively high inoculum sizes often lead to nutrient competition and metabolic burden, thereby weakening the long-term stability of the degradation process. Therefore, 1×10⁶ cells of *Hannaconia lactiflora* ZAFUHH1 were selected. 8 The concentration of cells / mL will be used for the next study. This concentration can maintain both high conversion rate and long-term metabolic activity.

[0056] 2. Effect of ZEN concentration on ZEN conversion by Hannah yeast ZAFU HH1 Add 1 mL of 1×10 8 A suspension of *Hannaconia solani* ZAFU HH1 cells / mL (prepared using the same method as above, hereinafter the same) was added to 50 mL of NYDB medium. An appropriate amount of ZEN standard solution was added to adjust the initial ZEN concentration to 2, 5, 10, 15, and 20 μg / mL. An equal volume of sterile water was used to replace 1 mL of 1×10⁻⁶ cells / mL of the ZEN standard solution. 8 A suspension of *Hannaconia henryi* ZAFU HH1 cells / mL was used as the control group for each treatment group. The cells were cultured at 28℃ and 150 rpm. Samples were collected at 0 and 36 hours of culture, and the residual ZEN concentration was determined by high-performance liquid chromatography (HPLC). ZEN conversion rate was calculated. Each group was repeated in triplicate.

[0057] The formula for calculating ZEN conversion rate is:

[0058] In the formula, φ ZEN conversion rate; S 0 represents the peak area of ​​the chromatogram of each group after 0 hours of ZEN conversion; S n The peak areas of the chromatograms of each group after 36 hours of ZEN conversion are given.

[0059] like Figure 3 As shown in Figure B (the ZEN conversion rate of the control groups in each treatment group was close to 0%, so it is not shown in the figure), when the initial ZEN concentration was 10, 15, and 20 μg / mL, the ZEN conversion rate dropped below 40%. When the initial ZEN concentration was 2 and 5 μg / mL, the ZEN conversion rate reached 70% or higher. To ensure a clearer evaluation of the conversion effect in subsequent experiments, 5 μg / mL was selected as the initial ZEN concentration.

[0060] Example 4: In vitro inhibitory effect of Hannahs yeast ZAFU HH1 on the growth of wild-type Fusarium graminearum strain PH-1 100 μL 1×108 A suspension of *H. hannaea* ZAFU HH1 cells / mL was placed in a blank culture dish, and 10 mL of PDA medium (after autoclaving but before the temperature reached solidification temperature) was added and mixed well to form the *H. hannaea* ZAFU HH1 treatment group (ZAFU HH1). 100 μL of sterile water was placed in a blank culture dish, and 10 mL of PDA medium (after autoclaving but before the temperature reached solidification temperature) was added and mixed well to form the control group (CK). Both were incubated at 28℃ for 24 hours. Then, another 10 mL of PDA medium (after autoclaving but before the temperature reached solidification temperature) was added to cover the top layer. After solidification, a *Fusarium graminearum* wild-type strain PH-1 mycelium block (7 mm in diameter) was placed in the center of the plate and incubated at 28℃ for observation. From day 3 to day 7, the diameter (mm) of the *Fusarium graminearum* wild-type strain PH-1 mycelium block in each group was measured daily. Each group had three replicates.

[0061] like Figure 4 As shown, compared with the control group, the Hannahs yeast ZAFU HH1 treatment group significantly inhibited the growth of Fusarium graminearum wild-type strain PH-1. Figure 4 A). After 7 days of incubation, the colony diameter in the control group was 89.33 mm, while the colony diameter in the Hannah yeast ZAFUHH1 treatment group was 23.33 mm. Figure 4 B). It is evident that Hannah's yeast ZAFU HH1 exhibits good in vitro inhibitory effects against Fusarium.

[0062] Example 5: Effect of actinomycete ketone (HMI) on the transformation of ZEN by Hannah yeast ZAFU HH1 Add 1 mL of 1×10 8 A suspension of *Hannaconia hannaensis* ZAFU HH1 cells / mL was inoculated into 50 mL of NYDB medium, and an appropriate amount of ZEN standard solution was added to adjust the initial ZEN concentration to 5 μg / mL. Three groups were set up: Group 1 (ZAFU HH1) received no actinomycete ketone, but an equal volume of sterile water; Group 2 (ZAFU HH1 + HMI) received 2.5 μg / mL actinomycete ketone (HMI) (the actinomycete ketone stock solution concentration was 1000 μg / mL, obtained by dissolving 10 mg of actinomycete ketone powder in 10 mL of methanol); Group 3 served as the control (CK), receiving no *Hannaconia hannaensis* ZAFU HH1 or actinomycete ketone, but an equal volume of sterile water. All groups were cultured at 28℃ and 150 rpm, and samples were collected at 0, 12, 24, 36, 48, and 60 hours. The residual ZEN concentration was determined using high-performance liquid chromatography (HPLC). Each group was replicated in triplicate.

[0063] like Figure 5As shown, the ZEN concentration in the sterile water control group did not decrease significantly within 60 hours. In both the ZAFUHH1 group without actinomycete ketone and the ZAFU HH1+HMI group with actinomycete ketone, the Hannah yeast ZAFU HH1 effectively converted ZEN in the culture medium after 36 hours of culture, indicating that actinomycete ketone has a limited inhibitory effect on the conversion of ZEN by Hannah yeast ZAFU HH1.

[0064] Example 6: Effect of different treatment solutions of Hannah yeast ZAFU HH1 on ZEN conversion efficiency 1. Live cells: Experimental group: 1 mL of 1×10 8 A suspension of *Hannaconia solani* ZAFU HH1 cells / mL was added to 50 mL of NYDB medium. One × 10⁻⁶ cells / mL of sterile water was used instead of 1 × 10⁻⁶ cells / mL. 8 A suspension of Hannah yeast ZAFU HH1 cells / mL was used as the control group.

[0065] 2. Heat-induced cell death: Experimental group: 1×10 8 A suspension of *Hannaconia solani* ZAFU HH1 cells / mL was heated in a 100 °C water bath for 30 minutes to kill the yeast. 1 mL of the heat-killed *Hannaconia solani* ZAFU HH1 suspension was added to 50 mL of NYDB medium. An equal volume of sterile water was used as a control group instead of the heat-killed *Hannaconia solani* ZAFU HH1 suspension.

[0066] 3. Extracellular substances: Experimental group: 1 mL of 1×10 8 A suspension of *Hannaconia solani* ZAFU HH1 cells / mL was added to 50 mL of NYDB medium and cultured at 28 °C and 150 rpm for 24 hours. The mixture was then centrifuged at 4 °C and 5000 g for 10 min, and the supernatant was collected. This supernatant was filtered through a 0.22 μm filter to obtain a cell-free filtrate. An equal volume of sterile water was used to replace 1×10⁻⁶ cells / mL of the filtrate. 8 A suspension of Hannah yeast ZAFU HH1 cells / mL was used as the control group.

[0067] 4. Intracellular substances: Experimental group: 1 mL of 1×10 8 A suspension of *Hannaconia solani* ZAFU HH1 cells / mL was added to 50 mL of NYDB medium and cultured at 28 °C and 150 rpm for 24 hours. The mixture was then centrifuged at 4 °C and 5000 g for 10 min, and the precipitate was collected. The precipitate was washed three times with PBS buffer (1X, pH 7.4; the same below), and then ground in liquid nitrogen. The ground powder was resuspended in 50 mL of PBS buffer, thoroughly mixed, and kept on ice for 30 minutes. The control group consisted of an equal volume of PBS buffer.

[0068] Each group has 3 replicates.

[0069] Add an appropriate amount of ZEN standard solution to each group to adjust the initial ZEN concentration to 5 μg / mL. Incubate at 28℃ and 150 rpm for 72 hours. Samples were collected at 0, 24, 36, 48, 60, and 72 hours during the incubation period. The residual ZEN concentration was determined using high-performance liquid chromatography (HPLC), and the ZEN conversion rate was calculated. The formula for calculating the ZEN conversion rate is:

[0070] In the formula, φ ZEN conversion rate; S 0 represents the peak area of ​​the chromatogram of each group after 0 hours of ZEN conversion (n=24, 36, 48, 60, 72). S n The peak areas of the chromatograms of ZEN converted to ZEN for n hours are given for each group (n=24, 36, 48, 60, 72).

[0071] like Figure 6 As shown (the ZEN conversion rate of the four control groups was close to 0%, so only the control group treated with live cells was used for plotting), heat-killed cells of *Saccharomyces hannaensis* ZAFU HH1 had almost no ZEN conversion effect, with a ZEN conversion rate of less than 5%. In contrast, the extracellular and intracellular substances of *Saccharomyces hannaensis* ZAFU HH1 showed ZEN conversion rates of 54.87% and 36.18% respectively at 36 hours, while the live cell ZEN conversion rate was the highest, reaching 85.87%. This result suggests that there may be a synergistic effect between the extracellular and intracellular substances of *Saccharomyces hannaensis* ZAFU HH1.

[0072] Example 7 Analysis and safety evaluation of ZEN products transformed with Hannah yeast ZAFU HH1 1. Product analysis based on UHPLC-Q-Orbitrap-HRMS 1 mL 1×10 8A suspension of *Hannaconia solani* ZAFU HH1 cells / mL was added to 50 mL of NYDB solution and cultured at 28°C and 150 rpm for 36 hours to obtain *Hannaconia solani* ZAFU HH1 culture medium. 500 μL of the culture medium was added to 500 μL of ethanol and centrifuged at 5000 g for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter. 20 mL of acetonitrile-water-acetic acid (70:29:1, v / v) was added to the filtered supernatant, vortexed for 1 min, and then shaken at 200 rpm for 30 min. The mixture was then centrifuged at 4000 rpm for 10 min to separate the solid and liquid. 0.5 mL of the supernatant was accurately transferred to a 1.5 mL centrifuge tube, diluted with 0.5 mL of water, vortexed for 1 min, and then centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter before analysis. Waters CORTECS™ UPLC C18 column (100 mm × 2.1 mm, 1.6 m); column temperature 40 °C; injection volume: 2 µL; mobile phase: A is methanol, B is an aqueous solution containing 0.1% (v / v) formic acid and 1 mmol / L ammonium acetate, flow rate: 0.3 mL / min; gradient elution conditions: 0–2 min, 90% B; 2–24 min, 90% B to 5% B; 24–27 min, 5% B; 27–30 min, 5% B to 90% B. The heated electrospray ionization source (HESI) temperature was 300℃; the capillary voltage was 3.2 kV; the ion transmission tube temperature was 320℃; the sheath gas was 35 units, and the auxiliary gas was 10 units. The full scan / ddms2 scanning mode had an acquisition range of 200–800 Da, with positive ion acquisition; the primary mass spectrometry resolution was 70,000 FWHM, and the secondary mass spectrometry resolution was 17,500 FWHM; the collision cell energy (NCE) was 20, 40, and 60 eV. like Figure 7-10 As shown, UHPLC-Q-Orbitrap-HRMS analysis revealed a retention time of 18.05 minutes for the ZEN parent compound, and detected its quasi-molecular ion peak [MH]. - m / z 317.1405 ( Figure 7 Two novel metabolites were identified after co-culturing Hannah yeast ZAFU HH1 with ZEN for 36 hours: product 1 (retention time 12.38 min) and product 2 (retention time 14.77 min), both exhibiting a quasi-molecular ion peak [MH]. - m / z 479.1944 ( Figure 8 and Figure 9The molecular weights of the two metabolites differ from ZEN (m / z 317.1405) by 162.0539 Da, a difference that corresponds to the theoretical increment of monosaccharide glycosylation (162.0528 Da, C6H). 10 The O5 group is highly compatible. Based on this, the molecular formula of the metabolite is inferred to be C. 24 H 32 O 10 The results are consistent with the characteristics of monoglucoside products. Based on the search results from the biotoxin database (Wu Yu, Ye Jin, Wang Songshan, Han Xiaomin, Li Li, Zhu Lin, Li Sen, Liu Hongmei, Wang Yang, Shao Yi, Wang Songxue. An accurate and efficient method for simultaneous determination of 16trichothecenes in maize and wheat by UHPLC-Q-Orbitrap-HRMS combined one-step pretreatment with a novel isotope multipoint calibration strategy[J]. FoodCotrol, 2023, 149.), compounds 1 and 2 are presumed to be ZEN-14-β-D-glucopyranoside and ZEN-16-β-D-glucopyranoside, respectively. Figure 10 ).

[0073] 2. Safety evaluation using Caenorhabditis elegans as a model (1) Effect of ZEN transformation product of Hannah yeast ZAFU HH1 on the number of Caenorhabditis elegans progeny Three groups were set up: a treatment group, a control group, and a positive control group. The treatment group received 1 mL of 1×10⁻⁶ solution. 8 A suspension of *Hannaconia hannaensis* ZAFU HH1 cells / mL was added to 50 mL of NYDB medium, and an appropriate amount of ZEN standard solution was added to adjust the initial ZEN concentration to 5 μg / mL. The medium was incubated at 28℃ and 150 rpm for 36 hours, followed by centrifugation at 4℃ and 5000 g for 10 min. The supernatant was collected and filtered through a 0.22 μm filter to obtain the culture filtrate of *Hannaconia hannaensis* ZAFU HH1 co-cultured with ZEN. The positive control group consisted of 0 hours of culture. The control group was prepared by replacing 1×10⁻⁶ cells / mL with an equal volume of sterile water. 8 Hannah yeast ZAFU HH1 suspension (cells / mL).

[0074] Synchronized *C. elegans* larvae (approximately 1000 larvae per mL of fluid) were transferred to NGM medium coated with inactivated *E. coli* OP50. A filtrate of *Hannaconia hannaea* ZAFU HH1 co-cultured with ZEN was added for chronic exposure, and the larvae were incubated at 20°C until adulthood. Adults were released into 96-well plates containing M9 buffer (200 μL M9 buffer per well, 1 adult larva per well), with 20 replicates per group. Egg counts at each stage were recorded under a microscope after 72 hours.

[0075] (2) Effect of ZEN transformation product of Hannahs yeast ZAFU HH1 on body length of Caenorhabditis elegans Three groups were set up: a treatment group, a control group, and a positive control group. The treatment group received 1 mL of 1×10⁻⁶ solution. 8 A suspension of *Hannaconia hannaensis* ZAFU HH1 cells / mL was added to 50 mL of NYDB medium, and an appropriate amount of ZEN standard solution was added to adjust the initial ZEN concentration to 5 μg / mL. The medium was incubated at 28℃ and 150 rpm for 36 hours, followed by centrifugation at 4℃ and 5000 g for 10 min. The supernatant was collected and filtered through a 0.22 μm filter to obtain the culture filtrate of *Hannaconia hannaensis* ZAFU HH1 co-cultured with ZEN. The positive control group consisted of 0 hours of culture. The control group was prepared by replacing 1×10⁻⁶ cells / mL with an equal volume of sterile water. 8 Hannah yeast ZAFU HH1 suspension (cells / mL).

[0076] Approximately 1000 L1-stage *C. elegans* worms (1 mL worm solution, approximately 1000 worms) were transferred to NGM medium coated with inactivated *E. coli* OP50. A filtrate of *Hannaconia hannagi* ZAFU HH1 co-cultured with ZEN was added for chronic exposure, and the worms were incubated at 20°C until adulthood. Body length was measured at each stage (L1, L2, L3, L4, and adult). Images were taken using a Nikon Eclipse Ni-E upright fluorescence microscope, and the worm images were analyzed using ImageJ software. Twenty replicates were performed per group.

[0077] like Figure 11 As shown, the average number of *C. elegans* progeny in the positive control group (ZEN not transformed with *H. hannaea* ZAFU HH1) decreased from 47.43 to 33.15 after 72 hours. In contrast, the number of progeny in the ZEN treatment group transformed with *H. hannaea* ZAFU HH1 was 45.69, which was not significantly different from the control group. Figure 11 A). This indicates that the product of ZEN transformed by Hannah's yeast ZAFUHH1 has a negligible effect on the number of progeny of Caenorhabditis elegans.

[0078] Both the positive control group and the treatment group exhibited developmental delays. From the L1 larval stage to the adult stage, the growth of the treatment group and the positive control group was significantly lagging behind that of the control group, with the most significant difference in adult body length. Figure 11 B). Compared with the control group, the body length of *C. elegans* in the early developmental stages of the treated groups showed little difference. At the L4 larval stage, the body length of the treated group was 474.80 μm, slightly shorter than the 480.77 μm of the control group. When *C. elegans* developed into adults, the average body length of the treated group was 877.84 μm, significantly higher than the 723.48 μm of the positive control group at the same developmental stage, but still lower than the 995.46 μm of the control group at the same developmental stage. Figure 11 C). These findings indicate that transformation with Hannah's yeast ZAFU HH1 delayed the growth inhibition of Caenorhabditis elegans by ZEN, but could not fully restore normal developmental parameters.

[0079] Example 8: Tolerance of Hannah's yeast ZAFU HH1 to temperature and acid stress Add 1 mL of 1×10 8 A suspension of *Hannaconia hannaensis* ZAFU HH1 cells / mL was added to 50 mL of NYDB medium prepared with citric acid at pH 4. The medium was incubated at 28 °C and 200 rpm. Culture media obtained after 0, 12, 24, 36, 48, and 60 hours of incubation were collected and diluted with sterile water to a final concentration of 1 × 10⁻⁶. 6 Cells / mL, take 100 μL and spread it on NYDA medium, incubate at 28 °C for 2 days, and then count the colonies. 8 Replace 1 mL of 1×10⁶ cells / mL Saccharomyces cerevisiae (laboratory model yeast) suspension. 8 A suspension of Hannah yeast ZAFU HH1 cells / mL was used as a control.

[0080] 1×10 8 A suspension of *Hannaconia solani* ZAFU HH1 cells / mL was incubated at 4°C, 28°C, and 45°C for 3 hours, followed by incubation at 25°C for 20 minutes to obtain *Hannaconia solani* ZAFU HH1 incubation solution. 100 μL of this incubation solution was plated onto NYDA medium and incubated at 28°C for 2 days, followed by colony counting. 8 Replace 1×10 cells / mL of Saccharomyces cerevisiae (laboratory model yeast) suspension. 8 A suspension of Hannah yeast ZAFUHH1 cells / mL was used as a control.

[0081] The subjects were divided into three treatment groups. The first treatment group (as a control) received 1 mL of 1×10⁻⁶ nitric acid solution in 50 mL of NYDB medium prepared with Na₂HPO₄ at pH 7. 8 A suspension of *Hannaconia solani* ZAFU HH1 cells / mL was prepared; an appropriate amount of ZEN standard solution was added to adjust the initial ZEN concentration to 5 μg / mL. The mixture was cultured at 28℃ and 150 rpm, and samples were collected at 0, 24, 36, 48, 60, and 72 hours. The residual ZEN concentration was determined using high-performance liquid chromatography (HPLC), and the ZEN conversion rate was calculated using the ZEN conversion rate calculation formula. The second treatment group differed from the first in that 50 mL of NYDB medium (pH 7) prepared with Na2HPO4 was replaced with 50 mL of NYDB medium (pH 4) prepared with citric acid. The third treatment group differed from the first in that it was cultured at 15℃ and 150 rpm. Each treatment group was treated with an equal volume of sterile water replacing 1 mL of 1×10⁻⁶ cells / mL of the ZEN standard solution. 8 A suspension of Hannah's yeast ZAFU HH1 cells / mL was used as the control group for this treatment group. Each group had 3 replicates.

[0082] The formula for calculating ZEN conversion rate is:

[0083] In the formula, φ ZEN conversion rate; S 0 represents the peak area of ​​the chromatogram of each group after 0 hours of ZEN conversion; S n The peak areas of the chromatograms of ZEN converted to ZEN for n hours are given for each group (n=24, 36, 48, 60, 72).

[0084] like Figure 12 As shown, compared to the laboratory-grown model yeast *Saccharomyces cerevisiae*, *Hannaconia ZAFU HH1* exhibits higher acid and low-temperature tolerance. Under acidic conditions (pH 4), the number of *Hannaconia ZAFU HH1* cells significantly exceeded that of *Saccharomyces cerevisiae* after approximately 18 hours of culture, rapidly multiplying to approximately 1.62 × 10⁻⁶ cells. 9 The CFU / mL concentration stabilized, and the acid tolerance of Hannah's yeast ZAFUHH1 was significantly better than that of Saccharomyces cerevisiae, demonstrating obvious acid resistance. Furthermore, it was found that after treatment at low temperature (4℃) for 3 hours, Hannah's yeast ZAFU HH1 could form 156 colonies on NYDA plates after 2 days of cultivation, significantly higher than the 89 colonies of Saccharomyces cerevisiae. This indicates that Hannah's yeast ZAFU HH1 exhibited significantly better low-temperature tolerance than Saccharomyces cerevisiae, demonstrating obvious low-temperature resistance.

[0085] Under acidic conditions (pH 4), Hannah yeast ZAFU HH1 exhibited a ZEN conversion rate of 47.10% after 36 hours. Figure 12 C, the ZEN conversion rate of the control groups in each treatment group was close to 0%, so it is not shown in the figure). Under low temperature conditions (15℃), Hannah yeast ZAFU HH1 showed a ZEN conversion rate of 57.48% after 36 hours. Figure 12 D. The ZEN conversion rate of the control groups in each treatment group was close to 0%, so it is not shown in the figure.

[0086] This invention discloses *Hannaconia hannaensis* ZAFU HH1, screened from soil in Tibet, which exhibits excellent ZEN conversion ability. During cultivation, it increases the ZEN conversion rate to 85.87% through extracellular metabolism and intracellular transformation. Notably, *Hannaconia hannaensis* ZAFU HH1 demonstrates excellent environmental adaptability: it grows well in acidic and low-temperature environments. This strain converts ZEN into the metabolites ZEN-14-β-D-glucopyranoside and ZEN-16-β-D-glucopyranoside, and its biosafety was verified using *C. elegans* experiments. These characteristics make *Hannaconia hannaensis* ZAFU HH1 an effective biological resource for ZEN control and also provide a strategic alternative for novel biotransformation technologies.

Claims

1. A strain of Hannazyme ZAFU HH1, its taxonomic name is... Hannaella zeae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 28, 2023, with accession number CGMCC No. 27725.

2. The bacterial agent prepared by Hannah's yeast ZAFU HH1 according to claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is prepared by the following method: Hannah yeast ZAFU HH1 is cultured in liquid to obtain a Hannah yeast ZAFU HH1 culture broth. The Hannah yeast ZAFU HH1 culture broth is centrifuged, and the bacterial cells are collected. The bacterial cells are washed with sterile water and then resuspended in sterile water to obtain the bacterial agent. The concentration of the bacterial agent is 1×10⁻⁶. 6 cells / mL -1×10 9 cells / mL.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent is prepared by the following method: Hannah yeast ZAFU HH1 is inoculated into a liquid culture medium, including NYDB medium, and cultured at 28-30℃ and 150-200 rpm for 1-3 days to obtain a Hannah yeast ZAFU HH1 culture broth. The Hannah yeast ZAFU HH1 culture broth is centrifuged to collect the bacterial cells. The bacterial cells are washed with sterile water and then resuspended in sterile water to obtain the bacterial agent, the concentration of which is 1×10⁻⁶. 6 cells / mL -1×10 9 cells / mL.

5. The application of Hannah's yeast ZAFU HH1 as described in claim 1 in the conversion of zearalenone.

6. The use of the microbial agent according to any one of claims 2-4 in the conversion of zearalenone.

7. The application of Hannah's yeast ZAFU HH1 according to claim 1 in inhibiting Fusarium, wherein the Fusarium is Fusarium graminearum.

8. The application of the microbial agent according to any one of claims 2-4 in inhibiting Fusarium, wherein the Fusarium is Fusarium graminearum.

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