Saccharomyces cerevisiae for degrading ochratoxin a and related application

Abstract

The present application belongs to the field of agricultural microorganism and plant technology, and particularly relates to a Saccharomyces cerevisiae for degrading ochratoxin A and related application. The Saccharomyces cerevisiae is Saccharomyces cerevisiae YC-YN, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC M 2026833. The strain is isolated from wine grape in the east foot of Helan Mountain, and can achieve a degradation rate of more than 90% on ochratoxin A within 48 hours. The optimal degradation condition is temperature of 28 DEG C, pH of 4.2, and inoculation amount of 5%. The present application further provides a microbial inoculant containing the strain and an application method of the microbial inoculant in degrading ochratoxin A. The constructed microbial inoculant has advantages of high efficiency, safety, wide applicable pH range, etc., and provides a new biological resource for green detoxification of ochratoxin A in food and feed, and is suitable for toxin reduction in the fields of grape wine, fruit juice, grain and feed, etc.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and plant technology, specifically relating to a brewer's yeast YC-YN that degrades ochratoxin A and its related applications. Background Technology

[0002] Saccharomyces cerevisiae is a single-celled fungus belonging to the genus Saccharomyces in the phylum Ascomycota. It is widely distributed in nature, especially on the surface of fruits such as grapes and in fermentation environments. Saccharomyces cerevisiae is characterized by rapid growth and reproduction, simple nutritional requirements, and strong acid and alcohol resistance, making it one of the most widely used microorganisms in the food fermentation industry. The cell wall of Saccharomyces cerevisiae has a certain adsorption capacity, and its metabolic activities can affect various trace components in the environment. In recent years, it has begun to attract attention in the field of food contaminant reduction.

[0003] Ochratoxin A (OTA) is a secondary metabolite produced by fungi of the genera *Penicillium* and *Aspergillus*. It exhibits nephrotoxicity, hepatotoxicity, immunotoxicity, and teratogenicity and carcinogenicity. OTA is widely found in grapes and grape products, grains, coffee, cocoa, and many other agricultural products. The International Agency for Research on Cancer (IARC) classifies it as a Group 2B carcinogen. The European Union sets the limit for OTA in wine at no more than 2 μg / kg, and many other countries have subsequently introduced strict limits. OTA contamination in grapes and wine is a major quality and safety issue facing the global wine industry. In some premium wine-producing regions, the climate is suitable for the growth of OTA-producing fungi, making OTA contamination control a key technological bottleneck for the industry's development.

[0004] Currently, the main methods for removing OTA (over-the-counter) substances include physical adsorption, chemical degradation, and biodegradation. Physical adsorption methods (such as those using activated carbon and bentonite) suffer from poor selectivity and the tendency to adsorb beneficial components easily; chemical degradation methods may introduce toxic byproducts, failing to meet food safety requirements. Biodegradation is considered the most promising strategy due to its green, safe, and efficient characteristics. However, most reported OTA-degrading microorganisms are bacteria or non-Saccharomyces cerevisiae strains, and their adaptability, tolerance (low pH, high alcohol, high sugar environments), and safety in actual wine fermentation environments limit their industrial application. Therefore, screening superior Saccharomyces cerevisiae strains with high OTA degradation capabilities from local fermentation environments and developing microbial resources that combine fermentation performance with detoxification functions is of great significance for the green and high-quality development of the wine industry. Summary of the Invention

[0005] This invention yielded a highly efficient OTA-degrading yeast strain, YC-YN, from Marselan wine grapes grown on the eastern foothills of the Helan Mountains in Ningxia. Molecular biological identification revealed that its ITS sequence accession number, MZ170795.1, showed less than 99.5% similarity to all previously reported OTA-degrading yeasts in public databases. This is the first time a native yeast with highly efficient OTA-degrading capabilities has been isolated from wine grapes grown on the eastern foothills of the Helan Mountains. Experiments showed that this strain achieved a 90.25% degradation rate of OTA at an initial concentration of 10 μg / L within 48 hours and exhibited excellent tolerance and degradation stability in a simulated grape juice fermentation system, providing a highly efficient native microbial resource for the biocontrol of OTA in winemaking.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a brewing yeast that degrades ochratoxin A. The brewing yeast is Saccharomyces cerevisiae YC-YN, which is deposited at the China Center for Type Culture Collection (CCTCCM) with accession number CCTCCM 2026833 and deposit date of April 27, 2026. The nucleotide sequence is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] .

[0010] The present invention also provides the application of the above-mentioned brewer's yeast YC-YN in the degradation of ochratoxin A.

[0011] The present invention also provides the application of the above-mentioned Saccharomyces cerevisiae YC-YN in the preparation of microbial agents for degrading ochratoxin A.

[0012] The present invention provides a microbial agent for degrading ochratoxin A, wherein the microbial agent comprises the above-mentioned brewer's yeast YC-YN.

[0013] In practical applications of this invention, the dosage forms of the microbial agent include fermentation broth and freeze-dried bacterial powder; the content of effective bacteria in the microbial agent is 1×10⁻⁶. 6 CFU / mL ~ 1×10 7 CFU / mL.

[0014] The present invention also provides a method for degrading ochratoxin A, the method comprising: mixing the above-mentioned brewer's yeast YC-YN or the above-mentioned microbial agent with the material to be treated; in the present invention, when brewer's yeast YC-YN is used, the inoculum amount of brewer's yeast YC-YN is 5% (V / V); when microbial agent is used, the ratio of microbial agent to material to be treated is 5mL:100mL; the treatment temperature is 28℃, the treatment pH value is 4.2; and the initial concentration of ochratoxin A in the material to be treated is 1~50μg / mL.

[0015] Beneficial effects:

[0016] (1) The brewing yeast YC-YN provided by the present invention was isolated from Marselan wine grapes on the eastern foothills of Helan Mountain in Ningxia. It is a native excellent strain of the production area. As can be seen from the contents recorded in the examples, it has natural adaptability and affinity to the natural environment and fermentation conditions of Ningxia wine production area, and can grow well and play a degradation role in the wine fermentation system.

[0017] (2) The strain of the present invention has excellent degradation ability for ochratoxin A. Under the condition of an initial concentration of 10 μg / L, the degradation rate reaches 90.25% within 48 hours, which is the highest degradation efficiency among the tested native brewing yeast strains, and is significantly better than the reported OTA-degrading yeast strains.

[0018] (3) The strain of the present invention has good tolerance to OTA and its growth is not significantly affected in the range of OTA concentration of 10 to 50 μg / L, indicating that it can maintain normal metabolic activity in OTA contamination environments of different degrees and has wide applicability;

[0019] (4) The strain of the present invention is brewing yeast, which is a recognized safe microorganism (GRAS) in the food industry. Compared with non-brewing yeast or bacterial strains, its application in the wine fermentation process does not require additional safety assessment and can be directly and seamlessly connected with the wine brewing process, which greatly reduces the technical threshold and approval cost for industrial application.

[0020] (5) The degradation activity of the strain of this invention is mainly located in intracellular enzymes, which can convert OTA into ochratoxin α, which has a much lower toxicity than OTA. The degradation products have been verified to have good safety by cytotoxicity experiments, and their degradation efficiency continues to increase during the 32-hour fermentation period. 600 The optimal degradation effect is achieved when the ratio is 1, which has good process adaptability and provides excellent strain resources for the development of safe and efficient OTA biocontrol technology for wine.

[0021] Biological Preservation Instructions

[0022] Saccharomyces cerevisiae YC-YN was deposited on April 27, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC M 2026833. Attached Figure Description

[0023] Figure 1 The growth curves of Saccharomyces cerevisiae YC-YN in YPD medium with different OTA contents are shown.

[0024] Figure 2 The figure shows the effects of different factors on the degradation of OTA by Saccharomyces cerevisiae YC-YN. The light color in the figure represents Saccharomyces cerevisiae YC-YN. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0026] Example

[0027] 1. Screening for strains that can degrade ochratoxin A

[0028] Using native Saccharomyces cerevisiae strains from Helan Mountain as the research object (strain information is shown in Table 1), we conducted a simulated grape juice fermentation experiment by activating the strains and adding ochratoxin A (OTA) exogenously. We preliminarily screened OTA-reducing strains and evaluated the OTA tolerance of the strains (10, 50 µg / L). We investigated the effects of different time (8, 16, 32, 48 h), temperature (15, 25, 25, 30 °C), and addition amount (0.4, 0.6, 0.8) on the removal capacity of the strains, explored the removal potential of OTA in simulated grape juice, and screened OTA-degrading strains.

[0029] Simulated grape juice preparation: Ratio: 100g / L glucose, 100g / L fructose, 2g / L ammonium sulfate, 5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, 0.2g / L sodium chloride and 0.05g / L manganese sulfate, diluted with water to volume, pH adjusted to 3.4 by tartaric acid.

[0030] Table 1. Strain Information

[0031] 1 MB-M-5-1 OQ876805.1 24 MB-C-2-2 OQ305020.1 2 2024-JM-167 OQ876799.1 25 MB-M-2-2 JQ824873.1 3 XM-C-2-11 OQ876799.1 26 XG-M-2-7 MG017562.1 4 LS-M-2-7 OQ876789.1 27 MB-C-2-12 MG017576.1 5 LS-C-5-1 MN462937.1 28 ZL-M-3-7 OL763537.1 6 2024-JM-199 OR786904.1 29 ZL-C-3-4 GU080046.1 7 HD-M-2-1 MG017584.1 30 ZL-C-1-5 OP644210.1 8 QTX11 MG017570.1 31 LS-C-4-5 OM037660.1 9 2024-JM-222 OM037663.1 32 XM-C-3-7 OP644147.1 10 2024-JM-212 MW989997.1 33 MH-C-4-4 OP604409.1 11 2024-JM-131 HM191660.1 34 MB-C-4-1 OR786921.1 12 2024-JM-224 MG017577.1 35 HD-M-5-1 OQ304802.1 13 ZL-C-5-2 MK907984.1 36 HD-C-4-1 HM101471.1 14 35B-NL KX428530.1 37 HD-M-4-2 OP281178.1 15 XG-C-3-2 EF192587.1 38 XG-M-4-3 OP281172.1 16 XG-M-3-2 KX428529.1 39 XM-M-4-4 LC389013.1 17 2024-JM-62 MG017580.1 40 XM-C-1-3 ​ 18 ​ ​ 41 ​ ​ 19 ​ ​ 42 ​ ​ 20 ​ ​ 43 ​ ​ 21 ​ ​ 44 ​ ​ 22 ​ ​ 45 ​ ​ 23 ​ ​ 46 ​ ​

[0032] 2. Validation of the UPLC-MS-MS method for the determination of ochratoxin A

[0033] The OTA determination method was evaluated based on precision and accuracy. Blank controls were set up, and three determinations were performed on different matrices (simulated grape juice, grapes, and wine) and different addition concentrations (1 µg / kg, 2 µg / kg, and 10 µg / kg) to verify the method (the results are shown in Table 2). The method recovery rate was 95%–111.3%, and the precision was 0.12%–4.6%, which met the requirements for accurate quantification.

[0034] Simulated grape juice formulation: 100g / L glucose, 100g / L fructose, 2g / L ammonium sulfate, 5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, 0.2g / L sodium chloride and 0.05g / L manganese sulfate, diluted to volume with water, and the pH value adjusted to 3.4 by tartaric acid.

[0035] The grapes used were commercially available; Marselan grapes were selected for this experiment.

[0036] The wine is a commercially available Spanish Golden Pigeon Select dry red wine (a blend of Cabernet Sauvignon and Marselan, 15% vol).

[0037] The extraction conditions for the sample were determined as follows: Weigh 5.00 g of simulated grape juice / grape / wine sample into a 50 mL centrifuge tube, add 20 mL of 80% acetonitrile solution, and sonicate for 30 min. Then, add 6 mL of the mixed purification material (1.0 g NaCl + 1.0 g MgSO4 + 0.2 g sodium citrate), shake immediately for 2 min, centrifuge at 4000 r / min for 5 min, collect 3 mL of the supernatant, dry it with N2, and make up to 1.5 mL with 50% acetonitrile solution. After vortexing, filter through a 0.22 μm filter membrane for analysis.

[0038] The chromatographic conditions were as follows: HSST3 column (2.1 mm × 100 mm, 1.8 μm); column temperature 40 ℃; injection volume 2 μL; flow rate 0.4 mL / min; mobile phase 0.1% formic acid methanol-5 mmol / L ammonium acetate 0.1% formic acid water; and gradient elution.

[0039] Mass spectrometry conditions were as follows: ion source: electrospray ionization source; scanning mode: negative ion mode; ion source spray voltage: 4500V for negative ion mode; ion source temperature: 550℃; spray gas flow rate: 50L / h; curtain gas flow rate: 50L / h; collision gas flow rate: 200–600L / h; external standard method for quantification. Scanning mode: multiple reaction monitoring (MRM).

[0040] Qualitative and quantitative methods: retention time 9.85, qualitative ion-pair charge-to-mass ratios 402.2 / 358.2 and 402.2 / 211.0, quantitative ion-pair charge-to-mass ratio 402.2 / 358.2, OTA external standard quantification.

[0041] Table 2. Accuracy and Precision of the Method

[0042]

[0043] 3. Comparison of the ability of yeast strains to remove ochratoxin A

[0044] 10 µg / L OTA was added exogenously, and the OTA content was measured in the sample taken on the 10th day after fermentation. The removal rate is shown in Table 3. The results show that the removal rate of YC-YN was 86.7%, which will be used as the yeast test material for subsequent studies.

[0045] Table 3 Evaluation of the effect of yeast in removing OTA

[0046] ​ ​ 7.25 ​ ​ 0 ​ ​ 0.25 ​ ​ 0 ​ ​ 0 ​ ​ 0 ​ ​ 3.25 ​ ​ 0 LS-C-5-1 MN462937.1 18 ZL-M-3-7 OL763537.1 0 2024-JM-199 OR786904.1 0 ZL-C-3-4 GU080046.1 0 HD-M-2-1 MG017584.1 4.5 ZL-C-1-5 OP644210.1 0 QTX11 MG017570.1 0 LS-C-4-5 OM037660.1 0 2024-JM-222 OM037663.1 0 XM-C-3-7 OP644147.1 0 2024-JM-212 MW989997.1 0 MH-C-4-4 OP604409.1 0 2024-JM-131 HM191660.1 0 MB-C-4-1 OR786921.1 0 2024-JM-224 MG017577.1 0 HD-M-5-1 OQ304802.1 0 ZL-C-5-2 MK907984.1 0 HD-C-4-1 HM101471.1 0 35B-NL KX428530.1 2.25 HD-M-4-2 OP281178.1 0 XG-C-3-2 EF192587.1 0 XG-M-4-3 OP281172.1 0 XG-M-3-2 KX428529.1 64.75 XM-M-4-4 LC389013.1 0 2024-JM-62 MG017580.1 0 XM-C-1-3 OR786906.1 0 MB-M-5-3 HM107798.1 2.25 LS-M-4-1 KX824758.1 10.75 YC21 MG017561.1 24.75 MH-C-5-3 KU729087.1 0 YC-XX LC413769.1 86.75 ZL-C-4-1 MZ768866.1 0 2024-JM-197 EU037089.1 0 MB-M-4-2 LC361430.1 0 YC14 (14D-NL) PP764050.1 11.25 LS-M-2-8 PP534168.1 0 YC-YN MZ170795.1 90.25 ZL-M-3-2 LC413770.1 0

[0047] 4. Analysis of yeast strains' tolerance to ochratoxin A

[0048] The yeast was activated in YPD medium and cultured on a shaker at 28°C and 150 rpm for 24 hours. 6 CFU / mL inoculum was inoculated into YPD medium with different OTA concentrations (0 μg / L, 10 μg / L, 50 μg / L) and incubated at 28℃ and 150 rpm for 48 h. Samples were taken at 0 h, 2 h, 8 h, 14 h, 24 h, and 48 h, and colony counts were recorded at different time points using the plate count method. The logarithmic colony counts were used to plot the corresponding yeast growth curves (e.g., ...). Figure 1 ).

[0049] The results showed that the overall biomass of YC-YN yeast first increased and then stabilized, and different OTA contents had no significant effect on yeast growth (p<0.05). The growth curves of the yeast were basically consistent, indicating that the two yeast strains selected in the experiment could grow normally in the medium containing OTA. Therefore, the experiment on the removal of OTA by yeast can continue.

[0050] 5. Effects of different factors on the degradation of OTA by strains

[0051] To evaluate the effects of different fermentation temperatures, fermentation times, and strain concentrations on OTA degradation, single-factor experiments were conducted. The experimental protocols are shown in Table 4. The cultures were incubated with shaking at 150 r / min for 48 h, and the OTA concentration was 10 μg / L. Each experiment was conducted in triplicate.

[0052] Table 4 Experimental protocols for different factors affecting the degradation of OTA (10 μg / L) by bacterial strains

[0053] Temperature / °C 15 25 30 35 Time / h 8 16 32 48 <![CDATA[Concentration / OD 600 > 0.4 0.6 0.8 1

[0054] The results are as follows Figure 2 As shown, Figure 2This indicates that culture temperature has no significant effect on OTA degradation rate. With increasing temperature, the removal rate did not change significantly. Strain concentration has a significant impact on OTA degradation; as the concentration increases, the degradation rate of OTA gradually increases. When OD... 600 When the value is 1, the degradation rate reaches a relatively high level of 89.7%. As time goes on, the degradation rate of OTA slowly increases, and after 32 hours, the degradation rate of OTA reaches a relatively high level.

[0055] 6. Identification of strains

[0056] (1) Morphological characteristics

[0057] On YPD plates, the colonies of Saccharomyces cerevisiae YC-YN appear milky white, round, with neat edges and a smooth, moist surface.

[0058] (2) Physiological and biochemical characteristics

[0059] Physiological and biochemical experiments were conducted on carbon source assimilation and nitrogen source utilization. Molecular biological identification: Genomic DNA was extracted from the strain, and its endogenous transcriptional spacer region sequence or 26S rDNA D1 / D2 region sequence was amplified. The sequencing results were compared for homology in databases such as GenBank, and a phylogenetic tree was constructed. The identification results showed that *Saccharomyces cerevisiae* YC-YN belongs to *Saccharomyces cerevisiae*, and its sequence similarity to the type strain is greater than 99%. The nucleotide sequence is shown in SEQ ID NO.1:

[0060] .

[0061] 7. Study on the degradation characteristics of the strain

[0062] (1) Degradation efficiency and kinetics

[0063] Under optimal culture conditions, Saccharomyces cerevisiae YC-YN exhibited high efficiency in degrading OTA at initial concentrations ranging from 1 to 50 μg / mL, with the fastest degradation rate occurring within 24 to 48 hours, consistent with a first-order kinetic model.

[0064] (2) Degradation Condition Optimization: The effects of temperature (20~40℃), pH (3.0~8.0), inoculum size, and initial OTA concentration on the degradation rate were studied. The optimal degradation conditions were determined to be: temperature 28℃, pH 4.2, and inoculum size 5%. Degradation Site Analysis: By comparing the degradation effects of intact cells, cell fragments, and sterile fermentation supernatant on OTA, it was determined that the degradation activity of Saccharomyces cerevisiae YC-YN is mainly located in intracellular enzymes. Degradation Product Analysis and Safety Evaluation: LC-MS / MS analysis of the degradation products revealed that OTA was mainly converted into the low-toxicity ochratoxin α (OTα, C11 H9O5Cl) and phenylalanine (C9H 11 NO2). Further cytotoxicity experiments (including human kidney cells HEK293) demonstrated that the toxicity of the degradation products was significantly reduced.

[0065] 8. Application of bacterial strains

[0066] Application in liquid systems: Activated brewer's yeast YC-YN cells or fermentation broth can be directly added to grape juice, wine, fermentation mash, etc., contaminated with OTA, and treated for a certain period of time under suitable conditions to effectively remove OTA.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A brewing yeast that degrades ochratoxin A, characterized in that, The brewing yeast is brewing yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae YC-YN, deposited at the China Center for Type Culture Collection, accession number CCTCCM 2026833, date of deposit: April 27, 2026.

2. The application of the brewing yeast YC-YN according to claim 1 in the degradation of ochratoxin A.

3. The application of the Saccharomyces cerevisiae YC-YN according to claim 1 in the preparation of microbial agents for degrading ochratoxin A.

4. A microbial agent for degrading ochratoxin A, characterized in that, The microbial agent includes the brewing yeast YC-YN as described in claim 1.

5. The microbial agent according to claim 4, characterized in that, The formulations of the microbial agents include fermentation broth and freeze-dried bacterial powder.

6. The microbial agent according to claim 4, characterized in that, The effective bacteria content in the microbial agent is 1×10⁻⁶. 6 CFU / mL ~ 1×10 8 CFU / mL.

7. A method for degrading ochratoxin A, characterized in that, The method includes: mixing the brewing yeast YC-YN of claim 1 or the microbial agent of claim 4 with the material to be treated.

8. The method according to claim 7, characterized in that, When using brewer's yeast YC-YN, the inoculum amount of brewer's yeast YC-YN is 5% by volume; When using microbial agents, the ratio of the microbial agents to the material to be treated is 5 mL: 100 mL.

9. The method according to claim 7, characterized in that, The treatment temperature was 28°C, and the pH value of the treatment was 4.

2.

10. The method according to claim 7, characterized in that, The initial concentration of ochratoxin A in the material to be treated is 1~50 μg / mL.

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