A method for glucosylating zearalenone and its derivatives

Abstract

The present invention discloses a method for glucosylating zearalenone and its derivatives. This method can efficiently convert more than 97% of the ZEN toxin in the reaction system into ZEN diglucoside; and for ZEN derivatives (ZAN, α-ZOL, β-ZOL), glucosylation reactions can also occur, with conversion rates ranging from 76% to 100%. Using this method, the conversion conditions are mild, the conversion method is simple, and the reaction time is short. Under the conditions of a reaction temperature of 30 °C and a shaker speed of 150 - 180 r / min, oscillating the reaction for 4 - 24 h can effectively convert the mycotoxin ZEN and its derivatives into less toxic or non-toxic products, achieving the green detoxification treatment of ZEN and its derivatives. It has advantages such as low cost, low process requirements, and simple operation for future industrial applications, has a wide range of applications and good conversion prospects, and has far-reaching significance for realizing the green detoxification treatment of ZEN and its derivatives.

Description

Technical Field

[0001] The invention belongs to the technical field of biodegradation, and in particular relates to a method for glucosidating zearalenone and its derivatives. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a non-steroidal estrogenic fungal toxin mainly produced by Fusarium. It is widely found in moldy corn, barley, wheat, sorghum and other grains and their by-products. ZEN enters the food chain through contaminated grain agricultural and sideline products and feed. After being absorbed by animals, it can easily cause infertility, miscarriage and stillbirth. It is also highly mutagenic and carcinogenic, seriously endangering livestock and human health.

[0003] In order to control ZEN pollution, countries around the world have set strict limits on the ZEN content in food: France stipulates that the ZEN content in cereals and vegetable oils cannot exceed 0.2mg / kg, and Russia stipulates that the content of rice, wheat, and flour cannot exceed 1mg / kg. In my country, the ZEN content in wheat and corn for human consumption cannot exceed 0.06mg / kg, and the ZEN content in feed cannot exceed 0.5mg / kg. However, from a global perspective, the ZEN pollution situation of food crops is not optimistic, which has a real and serious negative impact on the import and export of agricultural products. How to effectively detoxify has become a major social problem that needs to be solved urgently.

[0004] Biotransformation detoxification ZEN technology can avoid the shortcomings of physical and chemical methods such as limited detoxification effect, large nutrient loss, high cost and residual harmful substances, and has become a research hotspot and main trend. At present, the biological detoxification method mainly focuses on using ZEN as the only carbon source to screen degrading bacteria with degradation effect from contaminated soil and animal rumen, and then identify the strains through biochemical identification, and then separate and express the degradation enzyme and analyze the degradation products. There are the following disadvantages: 1) The screening workload is large, and the safety and toxicity of the degrading bacteria obtained by screening cannot be guaranteed, which is not conducive to the subsequent application in actual detoxification; 2) To a large extent, the degradation products of most of the screened degrading bacteria after acting on toxins are unknown, and some products are even more toxic than the parent toxins, which are considered to be ineffective detoxification. Among the main derivatives of ZEN, the toxicity is arranged as α-ZOL>α-ZAL (α-zearalanol)>β-ZAL (β-zearalanol)>ZAN>ZEN>β-ZOL. ZEN and its derivatives can produce synergistic effects and enhance toxicity, making the symptoms of poisoning more complicated. Therefore, there is an urgent need for a method to catalyze the conversion of ZEN and its derivatives into less toxic or non-toxic products, thereby promoting the further development and application of ZEN biodegradation technology in grains and feed. Summary of the invention

[0005] In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a method for glucosylating zearalenone and its derivatives. This method can efficiently convert more than 97% of the ZEN toxin (20 μg / mL) in the reaction system into ZEN diglucoside (ZEN-14,16-diG); and for the three main toxic derivatives of ZEN, namely ZAN (zearalanone), α-ZOL (α-zearalenol), and β-ZOL (β-zearalenol), glucosylation reactions can also occur, with a conversion rate of 76% to 100%.

[0006] Starting from the structural and toxicity analysis of numerous known ZEN degradation products and their derivatives, the present invention discovers that the glucosylated products of ZEN cannot bind to and activate estrogen receptors, and have advantages such as low toxicity and good stability (Dellafiora L, Ruotolo R, Perotti A, et al. Molecular insights on xenoestrogenic potential of zearalenone-14-glucoside through a mixed in vitro / in silico approach[J]. Food & Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association, 2017, 108(Pt A):257.).

[0007] Considering the future application costs and operational convenience requirements, after numerous screenings and repeated experimental adjustments of the catalytic conditions, the present invention proposes a method for glucosylating ZEN and its derivatives using Candida microorganism whole cells, which forms a favorable new supplement for the green removal of ZEN estrogen toxicity.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides an application of Candida in the whole cell catalysis of zearalenone and its derivatives glucosylation.

[0010] Furthermore, the substrate used for catalyzing the glucosylation of zearalenone and its derivatives is glucose.

[0011] Preferably, the Candida is at least one of Candida tropicalis and Candida parapsilosis;

[0012] Further, the Candida tropicalis is at least one of Candida tropicalis GIM2.147, Candida tropicalis CICC31949, Candida tropicalis CICC 1798, etc., but not limited thereto.

[0013] The Candida parapsilosis is at least one of Candida parapsilosis ATCC 7330, Candida parapsilosis CCTCC NO: M 2012491, Candida parapsilosis ACCC 20221, etc., but not limited thereto; among them, Candida parapsilosis CCTCC NO: M 2012491 is disclosed in the patent "CN201310081411.3, A Strain Producing Mannitol and a Method for Fermenting and Producing Mannitol with the Strain".

[0014] A method for glucosylating zearalenone and its derivatives, comprising the following steps:

[0015] (1) Take the Candida yeast seed liquid and inoculate it into the fermentation medium at an inoculation amount of 5% - 10% (v / v) respectively, and culture to obtain the fermentation broth;

[0016] (2) Take the fermentation broth obtained in step (1), centrifuge it at a low speed, discard the supernatant, wash it twice with sterile physiological saline, centrifuge it at a low speed, discard the supernatant, and collect the cell catalyst;

[0017] (3) Add the whole cell catalyst to the phosphate buffer solution containing ZEN and its derivatives at a dosage of 60 - 200 mg / mL, and then add glucose and oscillate for reaction.

[0018] Preferably, in step (1), the preparation method of the Candida yeast seed liquid comprises the following steps:

[0019] Take the Candida yeast strain, inoculate it into 20 - 50 mL of the seed medium, and culture it at 30 ± 3 °C and a shaking speed of 180 - 220 r / min for 12 - 16 h to obtain the seed liquid.

[0020] The formula of the seed medium: 30 g / L glucose, 6 g / L yeast extract, 6 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0021] Preferably, in step (1), the dosage of the fermentation medium is 50-100 mL.

[0022] Preferably, in step (1), the culturing conditions are culturing at 30±3 °C and 180-220 r / min for 20-24 h;

[0023] Preferably, in step (1), the formula of the fermentation medium: 46 g / L glucose, 12 g / L yeast extract, 3 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0024] Preferably, in step (2), the conditions of the low-speed centrifugation are 3500-4000×g, centrifuging at low temperature for 10-15 min; the low temperature is 4-6 °C;

[0025] Preferably, in step (2), the sterile normal saline is a NaCl solution with a concentration of 0.85%-0.9%, sterilized at 121 °C for 15 min.

[0026] Preferably, in step (3), the initial concentration of the ZEN and its derivatives is 10-40 μg / mL.

[0027] Preferably, in step (3), the molar concentration of the phosphate buffer solution is 0.01 mol / L, and the pH is 7.2-7.4.

[0028] Preferably, in step (3), the whole-cell catalyst is added to the phosphate buffer solution containing ZEN and its derivatives at a dosage of 100 mg / mL;

[0029] Furthermore, in step (3), the volume of the phosphate buffer solution containing ZEN and its derivatives is 5-20 mL;

[0030] Preferably, in step (3), the concentration of glucose is 5-20 mg / mL.

[0031] Preferably, in step (3), the conditions of the oscillating reaction are 30±3 °C, oscillating reaction at 150-180 r / min for 4-24 h.

[0032] Preferably, in step (3), the derivative is at least one of ZAN, α-ZOL, and β-ZOL.

[0033] The present invention has the following advantages and effects compared with the prior art:

[0034] (1) First of all, there are very few microorganisms reported that can catalyze glycosylation reactions. The references published include Bacillus cereus, Lactobacillus reuteri, Lactobacillus plantarum, molds, etc. There is no report on the glycosylation of ZEN by Candida. Moreover, there are many types of Candida. The discovery that Candida tropicalis and Candida parapsilosis are used as new whole-cell microorganisms to catalyze the glycosylation of ZEN is a major discovery by the inventors through repeated experiments and screening and comparative analysis of numerous strains; this not only effectively supplements the functions of the whole-cell microorganisms of Candida, but also provides a new research direction for the green detoxification technology of ZEN.

[0035] (2) Currently, the microorganisms reported to be able to catalyze the glycosylation of ZEN are only Thamnidium elegans, Rhizopus sp., Mucor bainieri, etc. The structures of the products of glycosylated ZEN by Thamnidium elegans (NRRL1613) and Mucor bainieri (NRRL 2988) have been published. They both catalyze the dehydration condensation of the hydroxyl group at the C14 or C16 position of ZEN with glucose to form monoglycosylated products ZEN-14-glucoside (ZEN-14-G) or ZEN-16-glucoside (ZEN-16-G). However, the glycosylation of ZEN by Candida described in the present invention is the simultaneous dehydration condensation of the hydroxyl groups at the C14 and C16 positions of ZEN with 2 molecules of glucose, and the product is ZEN-14,16-diG. When two glucose molecules bind to ZEN to form a glycosylated product and bind to the estrogen receptor, the glucose molecule first produces a weak interaction with the periphery of the estrogen receptor, and the large volume of the glycoside part hinders the channel for ZEN to enter the active center of the receptor, fundamentally affecting their binding. Compared with the monoglycosylated products ZEN-16-G and ZEN-14-G, the estrogen toxicity of ZEN-14,16-diG is weaker. This glycosylation method is more significant in terms of detoxification effect than the glycosylation of ZEN by other reported microorganisms.

[0036] (3) The glycosyl donors for catalytic glycosylation reactions reported in existing literature and patents are mainly UDP. For example, Saerens et al. used Candida bombicola ATCC 22214 to produce sophorolipids by fermentation glycosylation, using UDP as the glycosyl donor. The HvUGT14077 enzyme from barley also uses UDP as the glycosyl donor to catalyze the glycosylation of ZEN into ZEN-14G and ZEN-16G. However, the Candida - catalyzed glycosylation reaction described in this invention uses glucose as the glycosyl donor, which is significantly different from what is reported in other literature and patents; moreover, using glucose as the glycosyl donor can greatly reduce the reaction cost, which has far - reaching significance for industrial production applications.

[0037] (4) The method of the present invention has a high conversion efficiency. It can directionally catalyze more than 97% of ZEN (10 - 20 μg / mL) and its derivatives in the reaction system to be converted into diglucosylated products within 4 - 24 h, and the conversion rate of derivatives is 76 - 100%. This shows that the whole cells of Candida have the ability to efficiently glucosylate ZEN and its derivatives, which has not been reported in the prior art. Moreover, the directional catalysis method of Candida of the present invention also has very good effects on the derivatives of ZEN, which has not been reported in relevant literature and patents.

[0038] (5) Starting from the structural and toxicity analysis of numerous known ZEN degradation products and their derivatives, the present invention found that the whole cells of Candida can catalyze the glycosylation of ZEN and its derivatives. To obtain higher glycosylation efficiency, the inventor improved the formulations for Candida seed culture and fermentation culture respectively: on the basis of the conventional medium formulation, adjusted the addition amounts of glucose and yeast extract, and replaced the components of the amine salt. Through a large number of experiments and data analysis by the inventor, the ratio and addition amount were optimized, which is very beneficial to the growth of Candida and the improvement of glycosylation efficiency.

[0039] (6) The method of the present invention has mild conversion conditions, a simple conversion method, and a short reaction time. Under the conditions of a reaction temperature of 30 °C and a shaker speed of 150 - 180 r / min, oscillating for 4 - 24 h can effectively convert the mycotoxin ZEN and its derivatives into products with less toxicity or non - toxicity, realizing the green detoxification treatment of ZEN and its derivatives. It has advantages such as low cost, low process requirements, and simple operation for future industrial applications, has a wide application range and good conversion prospects, and has far - reaching significance for realizing the green detoxification treatment of ZEN and its derivatives. Description of the Drawings

[0040] Figure 1 It is the standard curve obtained by detecting the ZEN standard product by high - performance liquid chromatography in Example 1.

[0041] Figure 2 It is the liquid-phase detection chart of the ZEN conversion product after 4 h of conversion reaction using the whole-cell catalyst Candida tropicalis GIM2.147 in Example 1.

[0042] Figure 3 It is the 13 13C NMR spectrum of the product ZEN-14,16-diglucoside (ZEN-14,16-diG) of ZEN glucosylation in Example 1.

[0043] Figure 4 It is the conversion rate of ZEN and three derivatives β-ZOL, α-ZOL, and ZAN in the reaction system catalyzed by the whole-cell catalyst Candida tropicalis GIM2.147 for 4 h in Example 1.

[0044] Figure 5 It is the liquid-phase detection chart of the ZEN conversion product after the conversion reaction of the reaction system using the whole-cell catalyst Candida tropicalis CICC31949 in Example 2; among them, A: the liquid-phase detection chart of the reaction system after 0 h of reaction, B: the liquid-phase detection chart of the reaction system after 12 h of reaction.

[0045] Figure 6 It is the conversion rate of ZEN and three derivatives β-ZOL, α-ZOL, and ZAN in the reaction system catalyzed by the whole-cell catalyst Candida tropicalis CICC31949 for 12 h in Example 2.

[0046] Figure 7 It is the conversion rate of ZEN and three derivatives β-ZOL, α-ZOL, and ZAN in the reaction system catalyzed by the whole-cell catalyst Candida tropicalis CICC1798 for 24 h in Example 3.

[0047] Figure 8 It is the liquid-phase detection chart of the ZEN conversion product after 4 h of conversion reaction using the whole-cell catalyst Candida parapsilosis ATCC7330 in Example 4.

[0048] Figure 9 It is the 1 1H NMR spectrum of the product ZEN-14,16-diglucoside (ZEN-14,16-diG) of ZEN glucosylation in Example 4.

[0049] Figure 10It is the conversion rate of ZEN and three derivatives, β-ZOL, α-ZOL, and ZAN, in the reaction system catalyzed by the whole-cell catalyst Candida parapsilosis ATCC 7330 of Example 4 for 4 hours.

[0050] Figure 11 It is the liquid-phase detection chart of the conversion of three derivatives, β-ZOL, α-ZOL, and ZAN, of ZEN in the reaction system using the whole-cell catalyst Candida parapsilosis CCTCC NO: M 2012491 in Example 5; where a: the liquid-phase detection chart of β-ZOL at 0 h of the reaction; b: the liquid-phase detection chart of α-ZOL at 0 h of the reaction; c: the liquid-phase detection chart of ZAN at 0 h of the reaction; d: the liquid-phase detection chart of the conversion product of β-ZOL at 12 h of the reaction; e: the liquid-phase detection chart of the conversion product of α-ZOL at 12 h of the reaction; f: the liquid-phase detection chart of the conversion product of ZAN at 12 h of the reaction.

[0051] Figure 12 It is the conversion rate of ZEN and three derivatives, β-ZOL, α-ZOL, and ZAN, in the reaction system catalyzed by the whole-cell catalyst Candida parapsilosis CCTCC NO: M 2012491 in Example 5 for 12 hours.

[0052] Figure 13 It is the liquid-phase detection chart of the conversion of ZEN in the reaction system using the whole-cell catalyst Candida parapsilosis ACCC 20221 in Example 6; A: the liquid-phase detection chart of the reaction system after 0 h of the reaction, B: the liquid-phase detection chart of the reaction system after 12 h of the reaction.

[0053] Figure 14 It is the conversion rate of ZEN and three derivatives, β-ZOL, α-ZOL, and ZAN, in the reaction system catalyzed by the whole-cell catalyst Candida parapsilosis ACCC 20221 in Example 6 for 12 hours. Detailed implementation manners

[0054] The present invention will be further described in detail below in conjunction with the examples and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0055] For the test methods without specific experimental conditions noted in the following examples, they are generally in accordance with conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0056] Example 1 A method for glucosylating zearalenone and its derivatives, comprising the following steps:

[0057] (1) Preparation of fermentation broth: Take a loopful of the whole-cell catalyst of Candida tropicalis GIM2.147 (Guangdong Microbial Culture Collection Center) preserved in a slant tube, inoculate it into 50 mL of seed medium, and culture it at 30 °C and a shaking speed of 220 r / min for 16 h to obtain a seed solution. Inoculate it into 100 mL of fermentation medium at an inoculation amount of 5% (v / v) respectively, and culture it at 30 °C and 220 r / min for 20 h to obtain the fermentation broth.

[0058] The seed medium used is prepared as follows: Weigh 30 g of glucose, 6 g of yeast extract, 6 g of (NH 4 ) 2 SO 4 , 0.4 g of MgSO 4 ·7H 2 O, 1.0 g of KH 2 PO 4 , 1.0 g of K 2 HPO 4 , dissolve in 1000 mL of distilled water, and sterilize at 121 °C for 15 min.

[0059] The fermentation medium used is prepared as follows: Weigh 46 g of glucose, 12 g of yeast extract, 3 g of (NH 4 ) 2 SO 4 , 1 g of K 2 HPO 4 , 0.4 g of MgSO 4 ·7H 2 O and 1.0 g of KH 2 PO 4 , dissolve in 1000 mL of distilled water, and sterilize at 121 °C for 15 min.

[0060] (2) Take the fermentation broth obtained in step (1) and centrifuge it at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, add 10 mL of 0.85% sterile physiological saline and wash it twice, then centrifuge it at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, and obtain the wet cells, which are the whole-cell catalyst.

[0061] (3) Add 0.5 g of whole-cell catalyst to a phosphate buffer solution (1×, pH = 7.4) containing 20 μL of 5 mg / mL ZEN. The total volume of the reaction system is 5 mL, and the initial concentration of ZEN is 20 μg / mL. Finally, add 0.05 g of glucose and react for 4 h at 30 °C with a shaker speed of 180 r / min. Use the group without adding whole-cell catalyst as the control group and the experimental group without adding glucose as the positive control.

[0062] (4) Preparation of the ZEN standard curve: Dilute the ZEN stock solution (5 mg / mL) to working solutions with concentrations of 2, 4, 8, 16, 20, and 40 μg / mL respectively. Inject 20 μL and repeat the measurement three times and take the average. Draw the ZEN standard curve according to the relationship between the concentration and the peak area.

[0063] Prepare ZEN working solutions with concentrations of 2, 4, 8, 16, 20, and 40 μg / mL respectively, and measure the standard curve of the relationship between the ZEN concentration and the peak area. As Figure 1 shown, the correlation coefficient R 2 = 0.9981.

[0064] (5) Determination of the ZEN conversion rate by high-performance liquid chromatography:

[0065] After adding an equal volume of methanol to 0.5 mL of the reaction solution to stop the reaction, filter it through a 0.22 μm microporous filter membrane, and use HPLC with a UV detector to detect the content of ZEN in the degraded system and identify the degradation product β-ZOL by comparing with the standard product. The conditions for high-performance liquid chromatography to detect ZEN are as follows: Use a chromatographic column of Agilent ZORBAX SB-C18 (4.6 × 150 mm, 5 μm), and the mobile phase is acetonitrile / methanol / water at 46 / 8 / 46 (V / V / V). The column temperature during the test is 30 °C, the flow rate is 0.9 mL / min, and the wavelength of the detector is 237 nm. Calculate the conversion rate of ZEN using the following formula:

[0066]

[0067] The results of liquid-phase detection of ZEN are as Figure 2 shown, indicating that ZEN is rapidly converted into a new product after reacting for 4 h under the catalysis of Candida tropicalis GIM2.147. The product has a strong absorption peak at about 4 min, while the retention time of the original toxin ZEN is 19.76 min. The product may form a glycosylated structure due to the transfer of glucose molecules to the ZEN structure, resulting in an increase in the polarity of the product.

[0068] (6) Identification of the glucoside structure transformation product: React the reaction system prepared in step (3) at 30 °C with a shaker speed of 180 r / min for 4 h. After combining the reaction solutions, centrifuge at 8000×g for 5 min to remove the wet bacterial cells, add an equal volume of ethyl acetate for extraction 3 times, and obtain the crude product after vacuum distillation at 35 °C. Dissolve it in 1 mL of methanol, and after it is completely dissolved, spot it on a silica gel plate of GF 254 100×200 mm three times. Use ethyl acetate / ethanol (8 / 2) (v / v) as the chromatographic solvent. After thin-layer chromatography, observe the product and substrate bands through an ultraviolet lamp (254 nm). Collect the silica gel particles containing the product, dissolve the product with an appropriate amount of methanol, centrifuge at 4000×g for 5 min, take the supernatant, and dry it at 40 °C for 24 h to obtain the purified product. After purification, use deuterated dimethyl sulfoxide (DMSO) as the solvent, 600 MHz, and determine the structure of the product by 13 13C NMR, 1 1H NMR. The results are as shown in Figure 3 , indicating that in the glucoside structure of ZEN after glycosylation, the chemical shifts of C14 and C16 change significantly. Compared with the 13 13C NMR results of ZEN, the chemical shift of C14 moves from high field to low field by 1.36 ppm, and the chemical shift of C16 moves from high field to low field by 2.42 ppm. Moreover, in the 13 13C NMR of the product, chemical shifts from 61.9 ppm to 103.07 ppm are added (103.07 (C1`), 100.48 (C1``), 77.46 (C5`, C5``), 76.90 (C3`, C3``), 73.57 (C2`), 72.19 (C2``), 70.77 (C4`, C4``), 61.09 (C6`, C6``)), which do not exist in the carbon spectrum of the original toxin ZEN. It can be concluded that the glycosylation sites of the product should be on the hydroxyl groups of C14 and C16 on the benzene ring of ZEN.

[0069] (7) Add 1 g of the whole-cell catalyst to a phosphate buffer solution (1×, pH = 7.4) containing 200 μL of the derivative stock solution (1 mg / mL, ZAN, α-ZOL, β-ZOL). The total volume of the reaction system is 10 mL, and the initial concentration of the ZEN derivative is 20 μg / mL. Finally, add 0.1 g of glucose and react at 30 °C with a shaker speed of 180 r / min for 4 h. Use the group without adding the whole-cell catalyst as the control group, and use the experimental group without adding glucose as the positive control.

[0070] The method for detecting the conversion rate of the ZEN derivative is the same as that in steps (4) - (5) of this example.

[0071] The conversion rate results are as shown inFigure 4 As shown in the figure, it shows that Candida tropicalis GIM2.147 catalyzes the glycosylation of ZEN, and the conversion rate is 98% after 4 hours of reaction; under the same reaction conditions as ZEN, it also catalyzes the glycosylation of β-ZOL, α-ZOL and ZAN, and the conversion rates after 4 hours of reaction are 90%, 83% and 80% respectively.

[0072] Example 2 A method for glucosylating zearalenone and its derivatives, comprising the following steps:

[0073] (1) Preparation of fermentation broth: Take a loop of the preserved strain Candida tropicalis CICC 31949 (China Center for Industrial Culture Collection) from the slant tube, inoculate it into 40 mL of seed medium, and culture it at 30 °C and a shaking speed of 180 r / min for 12 h to obtain a seed solution. Inoculate it into 60 mL of fermentation medium at an inoculation amount of 5% (v / v) respectively, and culture it at 30 °C and 180 r / min for 24 h to obtain a fermentation broth.

[0074] The formula of the used seed medium is as follows: 30 g / L glucose, 6 g / L yeast extract, 6 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0075] The formula of the used fermentation medium is as follows: 46 g / L glucose, 12 g / L yeast extract, 3 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0076] (2) Take the fermentation broth from step (1) and centrifuge it at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, add 10 mL of 0.85% sterile physiological saline and wash it twice, centrifuge it at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, and obtain wet cells, which are the whole cell catalyst.

[0077] (3) Add 1 g of whole-cell catalyst to a phosphate buffer (1×, pH = 7.4) containing 20 μL of 5 mg / mL ZEN. The total volume of the reaction system is 10 mL, and the initial concentration of ZEN is 10 μg / mL. Finally, add 0.05 g of glucose, and react for 12 h at 30 °C with a shaker speed of 180 r / min. Use the group without adding whole-cell catalyst as the control group, and the experimental group without adding glucose as the positive control.

[0078] (4) Add 1 g of whole-cell catalyst to a phosphate buffer (1×, pH = 7.4) containing 200 μL of 1 mg / mL derivative stock solution (ZAN, α-ZOL, β-ZOL). The total volume of the reaction system is 10 mL, and the initial concentration of the ZEN derivative is 20 μg / mL. Finally, add 0.1 g of glucose, and react for 12 h at 30 °C with a shaker speed of 180 r / min. Use the group without adding whole-cell catalyst as the control group, and the experimental group without adding glucose as the positive control.

[0079] The methods for detecting the conversion rates of ZEN and its derivatives and verifying the product structures are the same as those in Example 1.

[0080] The results of liquid-phase detection of ZEN are as Figure 5 shown, indicating that ZEN is rapidly converted into the ZEN glucoside structure after 12 h of conversion reaction by the whole-cell catalyst of Candida tropicalis CICC 31949. The retention time of this product in the reversed-phase chromatography is 4.575 min.

[0081] The conversion rate results are as Figure 6 shown, indicating that the whole-cell catalyst of Candida tropicalis CICC 31949 catalyzes the glycosylation of ZEN, and the conversion rate after 12 h of reaction is 99%; under the same reaction conditions as ZEN, it also catalyzes the glycosylation of β-ZOL, α-ZOL, and ZAN, and the conversion rates after 12 h of reaction are 96%, 98%, and 99% respectively.

[0082] Example 3 A method for glucosylating zearalenone and its derivatives, comprising the following steps:

[0083] (1) Preparation of fermentation broth: Take a loop of the strain Candida tropicalis CICC 1798 (China Center for Industrial Culture Collection) preserved on a slant tube, inoculate it into 20 mL of seed medium, and culture it at 30 °C and a shaker speed of 180 r / min for 12 h to obtain a seed solution. Inoculate it into 50 mL of fermentation medium at an inoculation amount of 5% (v / v) respectively, and culture it at 30 °C and 180 r / min for 20 h to obtain the fermentation broth.

[0084] The formula of the seed culture medium used is as follows: 30 g / L glucose, 6 g / L yeast extract, 6 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0085] The formula of the fermentation culture medium used is as follows: 46 g / L glucose, 12 g / L yeast extract, 3 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0086] (2) Centrifuge the fermentation broth obtained in step (1) at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, add 10 mL of 0.85% sterile physiological saline and wash twice, then centrifuge at a low speed (3500×g, 4 °C) for 10 min, and discard the supernatant to obtain wet bacterial cells, which are the whole-cell catalyst.

[0087] (3) Add 0.5 g of the whole-cell catalyst to a phosphate buffer solution (1×, pH = 7.4) containing 20 μL of 5 mg / mL ZEN. The total volume of the reaction system is 5 mL, and the initial concentration of ZEN is 20 μg / mL. Finally, add 0.05 g of glucose and react at 30 °C with a shaker speed of 180 r / min for 24 h. Use the group without adding the whole-cell catalyst as the control group and the experimental group without adding glucose as the positive control.

[0088] (4) Add 1 g of the whole-cell catalyst to a phosphate buffer solution (1×, pH = 7.4) containing 200 μL of 1 mg / mL derivative stock solution (ZAN, α-ZOL, β-ZOL). The total volume of the reaction system is 10 mL, and the initial concentration of the ZEN derivative is 20 μg / mL. Finally, add 0.1 g of glucose and react at 30 °C with a shaker speed of 180 r / min for 24 h. Use the group without adding the whole-cell catalyst as the control group and the experimental group without adding glucose as the positive control.

[0089] Detect the conversion rates of catalyzing ZEN and its derivatives in the same way as in Example 1.

[0090] The conversion rate results are as Figure 7 shown, indicating that Candida tropicalis CICC 1798 catalyzes the glycosylation of ZEN, and the conversion rate is 91% after 24 h of reaction; under the same reaction conditions as ZEN, it also catalyzes the glycosylation of β-ZOL, α-ZOL, and ZAN, and the conversion rates are 77%, 84%, and 92% respectively after 24 h of reaction.

[0091] Example 4 A method for glycosylating zearalenone and its derivatives, comprising the following steps:

[0092] (1) Preparation of fermentation broth: Take a loop of the strain Candida parapsilosis ATCC 7330 preserved on a slant tube, inoculate it into 20 mL of seed medium, and culture it at 30 °C and a shaking speed of 180 r / min for 12 h to obtain a seed solution. Inoculate it into 50 mL of fermentation medium at an inoculation amount of 5% (v / v) and culture it at 30 °C and 180 r / min for 20 h to obtain a fermentation broth.

[0093] The formula of the used seed medium is as follows: 30 g / L glucose, 6 g / L yeast extract, 6 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0094] The formula of the used fermentation medium is as follows: 46 g / L glucose, 12 g / L yeast extract, 3 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0095] (2) Take the fermentation broth obtained in step (1) and centrifuge it at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, add 10 mL of 0.85% sterile physiological saline and wash it twice, then centrifuge it at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, and obtain wet cells, which are the whole-cell catalyst.

[0096] (3) Add 0.5 g of whole-cell catalyst to a phosphate buffer (1×, pH = 7.4) containing 20 μL of 5 mg / mL ZEN. The total volume of the reaction system is 5 mL, and the initial concentration of ZEN is 20 μg / mL. Finally, add 0.05 g of glucose and react for 4 h at 30 °C with a shaker speed of 180 r / min. Use the group without adding whole-cell catalyst as the control group and the experimental group without adding glucose as the positive control.

[0097] (4) Add 1 g of whole-cell catalyst to a phosphate buffer (1×, pH = 7.4) containing 200 μL of 1 mg / mL derivative stock solution (ZAN, α-ZOL, β-ZOL). The total volume of the reaction system is 10 mL, and the initial concentration of the ZEN derivative is 20 μg / mL. Finally, add 0.1 g of glucose and react for 4 h at 30 °C with a shaker speed of 180 r / min. Use the group without adding whole-cell catalyst as the control group and the experimental group without adding glucose as the positive control.

[0098] The method for detecting the conversion rate of ZEN and its derivatives is the same as that in Example 1.

[0099] The results of liquid-phase detection of ZEN are as Figure 8 shown, indicating that ZEN is rapidly converted into the glucoside structure of ZEN after reacting for 4 h under the catalysis of the whole-cell catalyst of Candida parapsilosis ATCC 7330. The retention time of this product in the reverse-phase chromatography is 4.148 min.

[0100] The identification method of the glucoside structure conversion product is the same as that in Example 1. 1 The results of 1H NMR are as Figure 9 shown, indicating that the chemical shift in the range of 3.26 ppm to 4.88 ppm is increased in the hydrogen spectrum (4.88 (d, J = 7.7 Hz, 1H), 4.70 (s, 1H), 3.69 (d, J = 11.7 Hz, 1H), 3.46 (s, 1H), 3.46 (d, J = 17.7 Hz, 0H), 3.43–3.26 (m, 4H)). It can be seen that there are significant chemical shift differences between the glycosylation product of ZEN under the catalysis of Candida parapsilosis ATCC 7330 and the original toxin ZEN.

[0101] The conversion rate results Figure 10As shown, it indicates that the whole-cell catalyst Candida parapsilosis ATCC 7330 of Candida parapsilosis catalyzes the glycosylation of ZEN, and the conversion rate is 87% after 4 hours of reaction; under the same reaction conditions as ZEN, it also catalyzes the glycosylation of β-ZOL, α-ZOL and ZAN, and the conversion rates after 4 hours of reaction are 77%, 76% and 83% respectively.

[0102] Example 5 A method for glycosylating zearalenone and its derivatives, comprising the following steps:

[0103] (1) Preparation of fermentation broth: Take a loop of the strain Candida parapsilosis CCTCC NO: M 2012491 preserved on a slant tube, inoculate it into 35 mL of seed medium, and culture it at 30 °C and a shaking speed of 200 r / min for 14 h to obtain a seed solution. Inoculate it into 70 mL of fermentation medium at an inoculation amount of 7% (v / v) respectively, and culture it at 30 °C and 200 r / min for 20 h to obtain fermentation broth.

[0104] The formula of the used seed medium is as follows: 30 g / L glucose, 6 g / L yeast extract, 6 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0105] The formula of the used fermentation medium is as follows: 46 g / L glucose, 12 g / L yeast extract, 3 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0106] (2) Take the fermentation broth in step (1) and centrifuge it at a low speed (3500×g, 4 °C) for 10 min, discard the supernatant, add 10 mL of 0.85% sterile physiological saline and wash it twice, centrifuge it at a low speed (3500×g, 4 °C) for 10 min, and discard the supernatant to obtain wet cells, which are the whole-cell catalyst.

[0107] (3) Add 0.5 g of whole-cell catalyst to a phosphate buffer solution (1×, pH = 7.4) containing 20 μL of 5 mg / mL ZEN. The total volume of the reaction system is 5 mL, and the initial concentration of ZEN is 20 μg / mL. Finally, add 0.05 g of glucose and react at 30 °C with a shaker speed of 180 r / min for 12 h. Use the group without adding the whole-cell catalyst as the control group, and use the experimental group without adding glucose as the positive control.

[0108] (4) Add 1 g of whole-cell catalyst to a phosphate buffer solution (1×, pH = 7.4) containing 200 μL of 1 mg / mL derivative stock solution (ZAN, α-ZOL, β-ZOL). The total volume of the reaction system is 10 mL, and the initial concentration of the ZEN derivative is 20 μg / mL. Finally, add 0.1 g of glucose and react at 30 °C with a shaker speed of 180 r / min for 12 h. Use the group without adding the whole-cell catalyst as the control group, and use the experimental group without adding glucose as the positive control.

[0109] The method for detecting the conversion rate of ZEN and its derivatives and verifying the product structure is the same as that in Example 1.

[0110] The results of liquid-phase detection of ZEN derivatives are as Figure 11 shown, indicating that the ZEN derivatives (β-ZOL, α-ZOL, ZAN) are rapidly converted into the glucoside structure of ZEN derivatives after reacting for 12 h under the catalysis of the whole-cell catalyst Candida parapsilosis CCTCC NO: M2012491. The retention times of these products in the reversed-phase chromatography are 3.133 min, 3.410 min, and 4.154 min, respectively.

[0111] The conversion rate results are as Figure 12 shown, indicating that Candida parapsilosis CCTCC NO: M 2012491 catalyzes the glycosylation of ZEN, and the conversion rate can reach 97% after reacting for 12 h; under the same reaction conditions as ZEN, it also catalyzes the glycosylation of β-ZOL, α-ZOL, and ZAN, and the conversion rates after reacting for 12 h are 98%, 76%, and 99%, respectively.

[0112] Example 6 A method for glucosylating zearalenone and its derivatives, comprising the following steps:

[0113] (1) Preparation of fermentation broth: One loop of the strain Candida parapsilosis ACCC 20221 (China Center for Agricultural Culture Collection) preserved on a slant tube was inoculated into 35 mL of seed medium and cultured at 30 °C with a shaking speed of 200 r / min for 14 h to obtain a seed solution. It was inoculated into 70 mL of fermentation medium at an inoculation amount of 7% (v / v) and cultured at 30 °C and 200 r / min for 20 h to obtain the fermentation broth.

[0114] The formula of the used seed medium is as follows: 30 g / L glucose, 6 g / L yeast extract, 6 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0115] The formula of the used fermentation medium is as follows: 46 g / L glucose, 12 g / L yeast extract, 3 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

[0116] (2) The fermentation broth obtained in step (1) was centrifuged at a low speed (3500×g, 4 °C) for 10 min, the supernatant was discarded, 10 mL of 0.85% sterile normal saline was added for washing twice, and then centrifuged at a low speed (3500×g, 4 °C) for 10 min, and the supernatant was discarded to obtain wet cells, which were the whole-cell catalyst.

[0117] (3) 0.5 g of the whole-cell catalyst was added to a phosphate buffer solution (1×, pH = 7.4) containing 40 μL of 5 mg / mL ZEN, and the total volume of the reaction system was 5 mL, where the initial concentration of ZEN was 40 μg / mL. Finally, 0.05 g of glucose was added, and the reaction was carried out at 30 °C with a shaking speed of 180 r / min for 12 h. The group without adding the whole-cell catalyst was used as the control group, and the experimental group without adding glucose was used as the positive control.

[0118] (4) Add 0.5 g of whole-cell catalyst to phosphate buffer (1×, pH = 7.4) containing 100 μL of 1 mg / mL derivative stock solution (ZAN, α-ZOL, β-ZOL). The total volume of the reaction system is 5 mL, and the initial concentration of ZEN derivative is 20 μg / mL. Finally, add 0.1 g of glucose and react for 12 h at 30 °C with a shaker speed of 180 r / min. Use the group without adding whole-cell catalyst as the control group and the experimental group without adding glucose as the positive control.

[0119] The method for detecting the conversion rate of ZEN and its derivatives and verifying the product structure is the same as that in Example 4.

[0120] The results of liquid-phase detection of ZEN are as Figure 13 shown, indicating that it is rapidly converted into the glucoside structure of ZEN after reacting for 12 h under the catalysis of Candida parapsilosis ACCC 20221. The retention time of this product in the reverse-phase chromatography is 4.497 min.

[0121] The conversion rate results are as Figure 14 shown, indicating that Candida parapsilosis ACCC20221 catalyzes the glycosylation of ZEN, and the conversion rate is 84% after reacting for 12 h; under the same reaction conditions as ZEN, it also catalyzes the glycosylation of β-ZOL, α-ZOL, and ZAN, and the conversion rates are 98%, 78%, and 87% respectively after reacting for 12 h.

[0122] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of Candida in the preparation of a product for whole-cell catalyzed glucosylation of zearalenone and its derivatives, Characterized in that: The Candida yeast is at least one of Candida tropicalis ( Candida tropicalis ) and Candida parapsilosis ( Candida parapsilosis ); The derivatives are at least one of zeranol, α-zearalenol and β-zearalenol; The substrate used for catalyzing the glucosylation of zearalenone and its derivatives is glucose.

2. The use according to claim 1, Characterized in that: The Candida tropicalis is at least one of Candida tropicalis GIM2.147, Candida tropicalis CICC 31949 and Candida tropicalis CICC 1798; The Candida parapsilosis is at least one of Candida parapsilosis ATCC 7330, Candida parapsilosis CCTCC NO: M2012491 and Candida parapsilosis ACCC 20221.

3. A method for glucosylating zearalenone and its derivatives, Characterized in that: Comprises the following steps: (1) Take the seed liquid of Candida in any one of claims 1 to 2, and inoculate it into the fermentation medium at an inoculation amount of 5% - 10% v / v respectively, and culture to obtain the fermentation broth; (2) Take the fermentation broth obtained in step (1), centrifuge it at a low speed, discard the supernatant, wash it twice with sterile normal saline, centrifuge it at a low speed, discard the supernatant, and collect the cell mass to obtain the whole-cell catalyst; (3) Add the whole-cell catalyst to the phosphate buffer solution containing zearalenone and its derivatives at a dosage of 60 - 200 mg / mL, and then add glucose, and oscillate for reaction; In step (3), the derivatives are at least one of zeranol, α-zearalenol and β-zearalenol.

4. The method for glucosylating zearalenone and its derivatives according to claim 3, Characterized in that: In step (3), the initial concentration of the zearalenone and its derivatives is 10 - 40 μg / mL; In step (3), the final concentration of glucose is 5 - 20 mg / mL.

5. The method for glucosylating zearalenone and its derivatives according to claim 3, Characterized in that: In step (3), the whole-cell catalyst is added to the phosphate buffer solution containing zearalenone and its derivatives at a dosage of 100 mg / mL; In step (3), the volume of the phosphate buffer solution containing zearalenone and its derivatives is 5 - 20 mL; In step (3), the conditions for the oscillating reaction are 30 ± 3°C, oscillating reaction at 150 - 180 r / min for 4 - 24 h.

6. The method for glucosylating zearalenone and its derivatives according to claim 3, Characterized in that: In step (1), the method for preparing the seed liquid of Candida comprises the following steps: Take the Candida strain, inoculate it into 20 - 50 mL of the seed medium, and culture it at 30 ± 3°C and a shaker speed of 180 - 220 r / min for 12 - 16 h to obtain the seed liquid; The formula of the seed culture medium: 30 g / L glucose, 6 g / L yeast extract, 6 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 ; In step (1), the dosage of the fermentation medium is 50 - 100 mL; In step (1), the conditions for the culture are culturing at 30 ± 3 °C and 180 - 220 r / min for 20 - 24 h; In step (1), the formula of the fermentation medium: 46 g / L glucose, 12 g / L yeast extract, 3 g / L (NH 4 ) 2 SO 4 , 1.0 g / L K 2 HPO 4 , 0.4 g / L MgSO 4 ·7H 2 O and 1.0 g / L KH 2 PO 4 .

7. According to the method for glucosylated zearalenone and its derivatives as claimed in claim 3, characterized in that: In step (2), the conditions for the low-speed centrifugation are 3500 - 4000 × g, centrifuging at low temperature for 10 - 15 min; the low temperature is 4 - 6 °C; In step (2), the sterile physiological saline is a NaCl solution with a concentration of 0.85% - 0.9%, sterilized at 121 °C for 15 min.

Images