Saccharomyces cerevisiae for reducing AFB1 and application thereof
Through the variable phthalassin strain SAU-AFT-D1 screened from Pixian bean paste, the problem of AFB1 contamination in fermented condiments was solved, and efficient and safe AFB1 removal effect was achieved.
Patent Information
- Application Number
- CN202510161524.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-13
- Publication Date
- 2025-06-17
AI Technical Summary
Mycotoxin AFB1 is often present in fermented condiments, and it is difficult to effectively remove the prior art without affecting the quality of the product.
The variable similiar yeast strain SAU-AFT-D1 was screened from Pixian bean paste. This strain has an AFB1 deduction rate of 88%, which can effectively reduce the AFB1 content without affecting the fermentation process.
This strain was able to deduct 20 ng/mL AFB1 in 24 hours to 88%, and maintained a high deducting efficiency under high concentration AFB1 environment, providing a safe and reliable method to remove AFB1.
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Figure CN120158381A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbial technology, and particularly relates to a strain of yeast for reducing AFB1 and its application. Background Art
[0002] The production of fermented condiments is a biochemical process based on the life activities of microorganisms. Various substances in the raw materials are decomposed and combined under the catalytic action of various enzymes produced by microorganisms to produce products containing various metabolites. Since the production process of fermented condiments requires the participation of microorganisms, it lays the foundation for being easily contaminated by mycotoxins, such as aflatoxin B1 (AFB1). Aflatoxin B1 is a mycotoxin that exists in grains, foods, and feeds, is more harmful to the human body and is more common, and the pollution situation is also the most serious. In the production process of fermented condiments, the change of the microbial flora is also related to the degree of mycotoxin pollution. Therefore, selecting strains that have the ability to inhibit the physiological activities of harmful fungi or reduce mycotoxins and do not affect the quality of fermented condiment products during the production process is currently an effective method to solve the problem of mycotoxin pollution in fermented condiments.
[0003] Yeasts are widely used in fermented products and are the main contributors to aroma and flavor substances. They can increase the content of ethyl acetate in the liquor fermentation process and increase the concentration of ester and alcohol flavor compounds. Yeast strains are commonly used in the fermented condiment manufacturing industry. There are relevant studies that the co-fermentation of Wickerhamomyces mutabilis with other strains can improve the flavor of fermented products. Currently, many microorganisms have been found to have the ability to degrade AFB1. For example, a strain Bacillus cereus XSSW9 was screened from Daqu and can degrade 80% of AFB1 with a final concentration of 1 mg / L. Lactobacillus plantarum The supernatant of FJS003 has the ability to degrade AFB1 and relies on the adsorption ability of the strain itself and inhibits the growth of Aspergillus flavus and Aspergillus fungi to reduce the probability of AFB1 persecution. Since the yeast in the present invention is screened from Pixian broad bean paste and has the ability to reduce AFB1, compared with the prior art, the present invention is the first to obtain Wickerhamomyces mutabilis with the ability to reduce AFB1 from Pixian broad bean paste. The source of this strain is safe and can provide a good bacterial source for reducing residual AFB1 in foods and fermented condiments. Summary of the Invention
[0004] The present invention relates to a strain of yeast for reducing AFB1 and its application.
[0005] The strain provided by the present invention is Wickerhamomyces mutabilis ( Wickerhamiella versatilis), the strain number is SAU-AFT-D1, which is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee. The deposit location is No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 32567. The deposit date is November 11, 2024.
[0006] The Wickerhamomyces mutabilis SAU-AFT-D1 was isolated and screened from Pixian broad bean paste. The intracellular enzyme of this strain achieved an AFB1 reduction rate of 88% at 20 ng / ml within 24 h. It can be applied to the reduction of residual AFB1 in foods and fermented condiments.
[0007] Adding 1-aminobenzotriazole (ABT) inhibits the reduction efficiency of the AFB1-reducing strain provided by the present invention. Under the inhibitory effect of ABT, the efficiency of reducing 40 ng / ml AFB1 by this strain decreased to 6.88% within 24 h.
[0008] The cultivation characteristics of the Wickerhamomyces mutabilis SAU-AFT-D1 are as follows: On a 3% NaCl YPD medium plate, after culturing at 30 °C for 5 d, the colonies were observed to be round, opaque, milky white, and viscous. The Wickerhamomyces mutabilis SAU-AFT-D1 was observed to have an oval shape and a budding reproduction process under 400× magnification. Description of the Drawings
[0009] Figure 1 A is the colony morphology diagram of the Wickerhamomyces mutabilis SAU-AFT-D1, Figure 1 B is the cell morphology diagram of the Wickerhamomyces mutabilis SAU-AFT-D1.
[0010] Figure 2 is the phylogenetic tree of the Wickerhamomyces mutabilis SAU-AFT-D1.
[0011] Figure 3 is the growth curve of the Wickerhamomyces mutabilis SAU-AFT-D1.
[0012] Figure 4 is the effect of substrate concentration on the reduction of AFB1 by the Wickerhamomyces mutabilis SAU-AFT-D1.
[0013] Figure 5 is the effect of ABT on the reduction of 40 ng / ml AFB1 by the Wickerhamomyces mutabilis SAU-AFT-D1. Detailed Embodiments
[0014] In the following examples, the test methods, unless otherwise specified, are all conventional methods; the reagents and biological materials, unless otherwise specified, can all be obtained from commercial channels; the percentage content, unless otherwise specified, is all mass percentage content.
[0015] The following examples further illustrate the specific implementation of the present invention, but are not limited to this example.
[0016] The formula of the 3% NaCl yeast complete medium (YPD) is as follows: 10 g of yeast extract (yeast leaching powder), 20 g of peptone, 20 g of anhydrous glucose, 30 g of NaCl, made up to 1000 mL with distilled water, and sterilized at 121 °C for 20 min. Adding 2% agar gives the solid medium.
[0017] Example 1: Identification and physiological and biochemical tests of Wickerhamomyces mutabilis SAU-AFT-D1.
[0018] The AFB1-reducing strain provided by the present invention was isolated and screened from Pixian broad bean paste and has good AFB1-reducing ability.
[0019] Morphological observation: Inoculate the target strain on a 3% NaCl YPD medium plate and observe the colony characteristics after culturing at 30 °C for 5 d; identify the colony characteristics and cell morphology with reference to the "Fungal Identification Manual". As Figure 1 shown in Figure A, the colony morphology of SAU-AFT-D1 is round, opaque, milky white, and viscous.
[0020] Cell morphology observation: Use the dilute iodine solution staining method. Drop a drop of dilute iodine solution on a clean glass slide, pick up a small amount of bacteria with an inoculation loop, place it in the staining solution, disperse the piled-up and overlapping bacteria in the staining solution with the inoculation loop, take a cover glass, make one side of the cover glass contact with the bacterial solution, and then slowly lower the cover glass to avoid generating bubbles, and observe under a microscope. As Figure 1 shown in Figure B, it was observed under 400× that the morphology of SAU-AFT-D1 is oval and there is a process of budding reproduction.
[0021] Molecular biology identification: DNA extraction was carried out by the kit method, using the extracted colony DNA as a template. ITS 1 and ITS 4 were used as primers for PCR amplification, and the PCR amplification products were sent to Sangon Biotech Co., Ltd. for sequencing.
[0022] Compare the sequence with the existing ITS sequences in NCBI for similarity analysis, select the sequences with high similarity to the test strain, and construct a phylogenetic tree with MEGA11. As Figure 2 shown, the sequence similarity of strain SAU-AFT-D1 with Wickerhamiella versatilis is as high as 99%. Combining the morphological results, SAU-AFT-D1 was determined to be Wickerhamomyces mutabilis.
[0023] Subtraction strain physiological and biochemical tests: The ability of the strain to ferment different sugars was tested using the sugar fermentation test. The single colony of the subtraction strain was inoculated into 3% NaCl YPD medium and cultured at 30 °C for 2 d to prepare a seed solution with a cell concentration of 1×10 8 CFU / mL. It was inoculated into the sugar fermentation medium at a ratio of v:v = 1:9 and cultured at 30 °C for 24 h, 48 h, and 6 d to observe the color change of the medium. Different sugars were selected: anhydrous glucose, sucrose, D-maltose, D-fructose, α-lactose monohydrate, L(+)-arabinose. A sterile 20% (m / v) sugar solution was added to the test tube at an addition amount of 5% (v / v). At the same time, the control groups were set up without inoculating the subtraction strain, without inoculating the strain, and without adding sugar. The strain SAU-AFT-D1 was able to ferment anhydrous glucose, sucrose, and D-maltose, and bubbles were observed in the Durham tubes. Acid was produced during the fermentation process, causing the pH of the medium to decrease, and the color of the medium changed from purple to yellow (Table 1).
[0024] Table 1 Acid production and gas production phenomena in the sugar fermentation experiment Group Time Acid production Gas production Time Acid production Gas production Time Acid production Gas production k - Sterile and sugar - free 24h - - 48h - - 6d - - k - Sugar - free 24h - - 48h - - 6d - - Anhydrous glucose 24h + - 48h + + 6d + + Sucrose 24h + - 48h + + 6d + + D - Maltose 24h + - 48h + + 6d + + D - Fructose 24h - - 48h - - 6d - + α - Lactose, monohydrate 24h - - 48h - - 6d - - L(+) Arabinose 24h - - 48h - - 6d - - Note: The color change phenomenon in the test tube indicates that the medium changes from alkaline to acidic, and acid is produced by yeast during fermentation. Yellow (+) is positive, light yellow and the liquid in the Durham tube is not purple, grayish yellow and there is light purple in the Durham tube, and remaining purple in the test tube are all negative (-); The gas production phenomenon is judged by the presence or absence of bubbles in the Durham tube, positive (+), negative (-).
[0025] Growth curve: Observe the growth trend of the strain in the AFB1 YPD medium with a final concentration of 100 ng / mL, and the group without adding AFB1 was used as the control. As Figure 3 shown, the growth curve of the strain SAU-AFT-D1 at a final concentration of 100 ng / mL AFB1 was similar to that of the control group, indicating that the strain can carry out normal physiological activities in the presence of AFB1.
[0026] Example 2: Subtraction characteristics of Wickerhamomyces mutabilis SAU-AFT-D1 against AFB1.
[0027] Effect of substrate concentration on the subtraction of AFB1 by SAU-AFT-D1: The seed solution of the strain SAU-AFT-D1 was inoculated into the YPD liquid medium containing 10, 20, 40, 60, 80, 100 ng / mL AFB1 (pH = 6.0) at an inoculation amount of 10% (v / v) and cultured at 30 °C for 24 h. High performance liquid chromatography (HPLC) was used to determine the subtraction rate of AFB1 by SAU-AFT-D1.
[0028] Effect of 1-aminobenzotriazole (ABT) on the reduction of AFB1 by SAU-AFT-D1: The seed liquid of strain SAU-AFT-D1 was inoculated into YPD liquid medium containing 40 ng / mL AFB1 (pH = 6.0) at an inoculation amount of 10% (v / v) and cultured at 30 °C for 24 h. ABT was not added to the control group, and 0.5 mM ABT was added to the experimental group for the experiment. High performance liquid chromatography was used to determine the reduction rate of AFB1 by SAU-AFT-D1.
[0029] Detection method for the reduction rate of AFB1 by SAU-AFT-D1: After collecting the fermentation broth, it was extracted 3 times with an equal volume of chloroform, dried under nitrogen at 40 °C, and finally dissolved in 10 mL of 50% (v / v) methanol to obtain the extract. The sample was purified using an AflaTest affinity column. Exactly 2.0 mL of the sample extract was accurately pipetted and made up to 20 mL with PBS buffer (pH 7.4) so that the methanol content did not exceed 15% (v / v). The immunoaffinity column was washed with PBS buffer and ultrapure water respectively, and the washing solution was discarded after emptying. After washing, exactly 2.0 mL of chromatographic grade methanol was accurately pipetted to elute the column twice, and the eluate was collected after emptying. Exactly 1.0 mL of the eluate (methanol eluate) was taken, dried under nitrogen at 40 °C, derivatized with 100 μL of trifluoroacetic acid for 20 min, dried by nitrogen, and then redissolved with a mixture of acetonitrile and water (v / v = 1 / 9), filtered through a 0.22 μm organic filter membrane and injected for detection. The filtrate was collected and the concentration of AFB1 was determined by high performance liquid chromatography, and the reduction rate of the strain to AFB1 was calculated.
[0030] The HPLC detection method is as follows: C18 chromatographic column (250 mm×4.6 mm, 5 μm), with acetonitrile: methanol: ultrapure water = 12:40:48 as the mobile phase, flow rate 0.8 mL / min, injection volume 20 μL, column oven temperature 40 °C, and the detector is a fluorescence detector, with excitation wavelength 360 nm and emission wavelength 450 nm.
[0031] The calculation formula for the reduction rate of AFB1 is:
[0032] A =(1 - S / C )*100%
[0033] In the formula: A is the reduction rate of AFB1 (%); S is the AFB1 concentration of the sample, C is the AFB1 concentration in the control group.
[0034] The reduction characteristics of strain SAU-AFT-D1 to AFB1 are as follows: As Figures 4 - 5As shown, the strain SAU-AFT-D1 exhibited the best reduction rate at 20 ng / mL. As the concentration of AFB1 increased, the reduction efficiency of this strain for AFB1 gradually decreased. However, when at a high concentration of 100 ng / mL, SAU-AFT-D1 still had a reduction rate of 14%. ABT inhibited the reduction ability of the AFB1-reducing strain, and the reduction rate for 40 ng / mL AFB1 decreased to 6.88% after 24 hours. The results of this study provide a data reference for the application of the strain SAU-AFT-D1 in foods and fermented condiments to reduce residual AFB1 therein.
[0035] The above examples merely describe the implementation methods of the present invention, rather than limiting the scope of the present invention. For those skilled in the art, the above description can be improved or modified, but all such improvements or modifications should fall within the protection scope determined by the claims of the present invention. Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a strain of Wickerhamomyces anomalus ( Wickerhamiella versatilis ) SAU-AFT-D1 that can effectively reduce AFB1 and its application.
Claims
1. A strain of Pseudomonas aeruginosa with a preservation number of CGMCC No. 32567 ( Wickerhamiella versatilis ) SAU-AFT-D1 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 11, 2024, and the deposit location is No. 1 Beichen West Road, Chaoyang District, Beijing.
2. according to claim 1 described Pseudomonas aeruginosa ( Wickerhamiella versatilis )SAU-AFT-D1, characterized by: The colony morphology is round, opaque, milky white, sticky, yeast, and the cells are oval.
3. according to claim 1 described Pseudomonas aeruginosa ( Wickerhamiella versatilis )SAU-AFT-D1, characterized by: The intracellular enzyme of the strain removed 20 ng / ml AFB1 at a rate of 88% in 24 h. The addition of 0.5 mM 1-aminobenzotriazole (ABT) reduced the strain's ability to remove 40 ng / mL AFB1 to 6.88% in 24 h.
4. The method according to claim 1 wherein the Pseudomonas aeruginosa ( Wickerhamiella versatilis )SAU-AFT-D1, characterized by: It provides a good bacterial source for removing residual AFB1 in food and fermented condiments.