Use of Saccharomyces cerevisiae Hansen isolated from China for degrading patulin

Abstract

The present invention belongs to the technical field of toxin degradation, and specifically relates to the use of Saccharomyces cerevisiae Hansen for degrading patulin; the steps are as follows: First, Saccharomyces cerevisiae Hansen is inoculated into NYDB medium. After culturing and activation, the bacterial liquid is spread on NYDA medium; then, single colonies are selected and inoculated into NYDB medium. After culturing, the cells are obtained by centrifugation. After washing with sterile distilled water, they are diluted into a bacterial suspension of 1×10<supgt;8< / supgt; cells / mL and inoculated into NYDB medium for culture. A patulin standard solution is added to the NYDB medium, and its initial concentration is adjusted to 10 μg / mL. Under light-shielded conditions, shake culture is carried out to achieve the use of degrading patulin; the results show that the degradation rate is close to 50% at 12 h, and complete degradation is achieved after 30 h, and patulin can be degraded efficiently and rapidly; moreover, the present invention is safe, environmentally friendly, harmless to humans, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of toxin degradation, and particularly relates to the use of Saccharomyces cerevisiae Hansen for degrading patulin. Background Art

[0002] Patulin, also known as patulin, is a highly toxic polyketide secondary metabolite with the chemical formula C 7 H 6 O 4 , with a molecular weight of 154.13. Patulin is a white crystal with a melting point of 110.5 - 112 °C. It is easily soluble in water with low pH and organic solvents such as ethanol, acetone, ethyl acetate, and chloroform. Patulin in an aqueous solution with a pH of 3.3 - 5.5 is relatively stable even in a high-temperature environment of 105 °C - 125 °C, while it is unstable under alkaline conditions. Its maximum ultraviolet absorption wavelength is 276 nm, and patulin can withstand high temperatures of 105 - 125 °C.

[0003] Among Penicillium, 13 species of fungi can produce patulin, and Penicillium expansum is the main producer of patulin, and its optimal temperature for producing patulin is 20 - 25 °C. In the early 20th century, patulin was reported as a broad-spectrum antibiotic because it could inhibit more than 75 different bacteria. Further clinical studies found that patulin has toxic effects on fungi and animal and plant cells. The toxicity of patulin is mainly manifested as acute toxicity, chronic toxicity, and cytotoxicity. At the same time, it is also a potential promoter of cancer development. Given the strong toxicity of patulin to humans and animals, many countries or organizations such as the United States, the European Union, and China have established strict limit standards for the content of patulin in food. China has also aligned with international standards and has established strict limit standards for the content of patulin in apples, hawthorns, and their products.

[0004] Currently, the methods for controlling patulin contamination mainly include physical methods, chemical methods, and biological methods. Physical methods mainly use measures such as manual selection, high-pressure water washing, refrigeration, filtration and adsorption, pasteurization, and radiation to treat patulin in products. However, physical methods require a large amount of manpower and material resources, with high costs, and some methods will affect product quality. Chemical methods for controlling patulin are mainly divided into two types: one is to kill pathogenic bacteria from the source through chemical fungicides to prevent patulin contamination; the other is to remove the already produced patulin by adding chemical substances. However, adding chemical substances requires in-depth understanding of their reaction mechanisms, whether the reaction products are toxic, etc., and some chemical substances will seriously damage product quality, resulting in the loss of nutrition and product value.

[0005] Biological control methods refer to using active antagonistic bacteria (yeasts) to inhibit the growth of pathogenic bacteria, thereby controlling the production of patulin, or directly removing the already produced patulin through microbial fermentation. In recent years, using microorganisms and their enzymes to degrade toxins has become a research hotspot. Although existing yeasts, lactic acid bacteria, and molds have been found to have the effect of degrading patulin, they face problems such as poor effect and slow degradation. Moreover, there are few yeasts that can degrade patulin currently, and there is no relevant report on Hannaella sinensis controlling patulin in China. Summary of the Invention

[0006] Aiming at the defects and deficiencies of the prior art, the present invention provides a strain of Hannaella sinensis isolated from the soil of an ecological orchard. Based on the degradation of patulin by Hannaella sinensis, it has high safety and extremely high degradation effect.

[0007] The strain of Hannaella sinensis for degrading patulin provided by the present invention was screened and isolated from the soil of the Shiyezhou Agricultural Ecological Orchard in Zhenjiang City. It was cultured at 28 °C on a NYDA solid medium plate for morphological observation. The sequence analysis of the 26S rRNA D1 / D2 region of the strain was carried out for molecular biological identification. It was identified as Hannaella sinensis YD, and its safety has been proven through an oral toxicity test on ICR mice, and it is harmless to humans. At the same time, the strain of Hannaella sinensis for degrading patulin provided by the present invention is currently preserved in the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. The preservation date is July 5, 2021, and the preservation number is: CCTCC NO: M2021821. The proposed taxonomic name is Hannaella sinensis YD. In addition, the strain used in the present invention has been applied for and publicly disclosed a Chinese invention patent, and the invention name is: A yeast strain for controlling post-harvest diseases of apples and its uses.

[0008] In order to achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0009] (1) Prepare a bacterial suspension: Inoculate Hannaella sinensis into NYDB medium and culture it at 25 - 28 °C under shaking conditions of 180 rpm for 20 - 24 h for activation to obtain the activated bacterial liquid. Then, spread the activated bacterial liquid on NYDA solid medium, and culture it at a temperature of 25 - 28 °C for 48 - 72 h. Then, select the single colony of Hannaella sinensis growing on NYDA solid medium and inoculate it into NYDB medium for culture. After culture, centrifuge to obtain the bacterial cells, wash them several times with sterile distilled water, centrifuge again after washing, and collect the bacterial cells and dilute them with sterile water to a bacterial suspension of 1×10 8 cells / mL;

[0010] (2) Take the bacterial suspension prepared in step (1), inoculate it into the NYDB medium, and then add the patulin standard solution to the NYDB medium. Under a light-proof environment, shake and culture to achieve the use of rapidly degrading patulin.

[0011] Preferably, in step (1), the rotation speed of the centrifugation is 7000 r / min, the time is 10 min, and the temperature is 4 °C.

[0012] Preferably, the components of the NYDA medium in step (1) are as follows: based on 1000 mL, 8 g of beef extract, 5 g of yeast extract, 10 g of glucose, 20 g of agar, and the balance is distilled water. Sterilize at 115 °C for 15 min by moist heat.

[0013] Preferably, the components of the NYDB medium in step (1) are as follows: based on 1000 mL, 5 g of yeast extract, 10 g of glucose, 8 g of beef extract, and the balance is distilled water. The pH is natural. Sterilize at 115 °C for 15 min.

[0014] Preferably, the temperature for culturing the single colony inoculated into the NYDB medium in step (1) is 25 °C to 28 °C, and the time is 48 h.

[0015] Preferably, the several times of washing with sterile distilled water in step (1) is specifically 2 to 3 times.

[0016] Preferably, the inoculation amount of the bacterial suspension inoculated into the NYDB medium in step (2) is 1% to 2%; that is, the bacterial suspension is 1% to 2% of the volume of the NYDB medium.

[0017] Preferably, the final concentration of patulin standard in the NYDB medium in step (2) is 10 μg / mL.

[0018] Preferably, the conditions for the shaking culture in step (2) are: 25 °C, 180 rpm, and the time is 12 to 30 h.

[0019] Advantages of the present invention:

[0020] (1) The intracellular enzyme of the Saccharomyces hanna in the present invention can efficiently and rapidly degrade patulin; when the initial concentration of PAT is 10 μg / mL, the degradation rate is close to 50% in 12 h, about 80% in 18 h, and complete degradation is achieved after 30 h, and the degradation effect is very remarkable.

[0021] (2) The Saccharomyces hanna used in the present invention is determined to be highly safe and harmless to humans through the mouse acute toxicity test. Therefore, it can be applied to control the patulin contamination in foods, feeds and their products, etc., and ensure the edible safety of foods, feeds and their products, etc. Brief Description of the Drawings

[0022] Figure 1 It is the degradation effect diagram of patulin by Saccharomyces hanna in China; among them: CK: the control group treated with sterile water; Y: the experimental group treated with Saccharomyces hanna in China. Detailed Implementation Modes

[0023] The present invention will be described in more detail by means of the following implementation examples; the following examples are only illustrative, and the present invention is not limited by these examples.

[0024] Example 1:

[0025] (1) Prepare the bacterial suspension: Inoculate Saccharomyces hanna in China into the NYDB medium, and culture it at 25 °C under the shaking condition of 180 rpm for 24 h for activation to obtain the activated bacterial liquid; then take the activated bacterial liquid and spread it on the NYDA solid medium, and culture it at 25 °C for 48 h; then, select 2 loops of single colonies of Saccharomyces hanna in China growing on the NYDA solid medium, inoculate them into 50 mL of PM medium for culture, and after centrifugation, the bacterial cells are obtained, washed 3 times with sterile distilled water, centrifuged again after washing, and the collected bacterial cells are diluted with sterile water to 1×10 8 cells / mL of bacterial suspension;

[0026] (2) Take the bacterial suspension prepared in step (1), inoculate it into the NYDB medium according to an addition amount of 1%, and then add the patulin standard solution to make its concentration in the NYDB medium 10 μg / mL, and culture it in the dark under the shaking condition of 25 °C and 180 rpm;

[0027] Sampling and detection: Starting from 0 h, sample every 6 h, centrifuge at 7000×g for 10 min, and then filter with a 0.22 μm organic filter membrane to measure the degradation effect of patulin;

[0028] Control group: The operation of the control group is the same as that of step (2) of Example 1, except that the bacterial suspension in step (2) is replaced with sterile water.

[0029] Determine its patulin content by HPLC: Determine the patulin content by high performance liquid chromatography. The liquid phase system is Agilent 1260 (Agilent Technologies, USA), and the chromatographic column is Zorbax SB-C 18 column (250 mm×4.6 mm, 5 μm, Agilent). The mobile phase is water and acetonitrile (90:10, V / V), the flow rate is 1.0 mL / min, the detection temperature is 28 °C, the injection volume is 20 μL, the detection time is 15 min, and the detection wavelength is 276 nm.

[0030] The detection results are as follows Figure 1 shown. Saccharomyces cerevisiae Hansen has an obvious degradation effect on patulin. When the initial concentration of PAT is 10 μg / mL, the degradation rate approaches 50% in 12 h, and exceeds 80% in 18 h. After culturing for 30 h, the concentration of patulin in the NYDB medium inoculated with Saccharomyces cerevisiae Hansen drops to the lowest detection limit of the instrument. However, the concentration of patulin in the control group does not decrease significantly within 30 h; this indicates that Saccharomyces cerevisiae Hansen cells play an important role in the degradation of patulin and can rapidly degrade patulin.

[0031] In summary, Saccharomyces cerevisiae Hansen has a significant degradation effect on patulin, and the bacterial suspension or biocontrol agent prepared therefrom can be widely used for the degradation of patulin in the fields of fruits, vegetables or fruit juices.

[0032] Note: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although the present specification has described the present invention in detail with reference to the above respective embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered within the scope of the claims of the present invention.

Claims

1. Use of Saccharomyces cerevisiae Hansen from China for degrading patulin, characterized in that, The preservation number of the **Hanseniaspora guilliermondii** is: CCTCC NO: M 2021821, and the **Hanseniaspora guilliermondii** used for degrading patulin is a bacterial suspension of 1×10 8 cells / mL.

2. The use according to claim 1, characterized in that, The steps for preparing the bacterial suspension are as follows: Inoculate **Hansenula sinensis** into NYDB medium, and culture it at 25-28 °C under shaking conditions of 180 rpm for 20-24 h for activation to obtain the activated bacterial liquid. Spread the activated bacterial liquid on NYDA solid medium, and culture it at 25-28 °C for 48-72 h. Then, select the single colony of **Hansenula sinensis** growing on NYDA solid medium and inoculate it into NYDB medium for culture. After culture, centrifuge to obtain the thalli, wash them several times with sterile distilled water, centrifuge again after washing, and collect the thalli and dilute them with sterile water to a bacterial suspension of 1×10 8 cells / mL.

3. The use according to claim 2, characterized in that, The rotation speed of the centrifugation is 7000 r / min, the time is 10 min, and the temperature is 4°C.

4. The use according to claim 2, characterized in that, The components of the NYDA medium are as follows: based on 1000 mL, 8 g of beef extract, 5 g of yeast extract, 10 g of glucose, 20 g of agar, and the balance is distilled water, sterilized by moist heat at 115°C for 15 min.

5. The use according to claim 2, characterized in that, The components of the NYDB medium are as follows: based on 1000 mL, 5 g of yeast extract, 10 g of glucose, 8 g of beef extract, and the balance is distilled water, with natural pH, sterilized at 115°C for 15 min.

6. The use according to claim 2, characterized in that, The temperature for culturing the single colony inoculated into the NYDB medium is 25°C to 28°C, and the time is 48 h.

7. The use according to claim 2, characterized in that, The specific number of times of washing with sterile distilled water is 2 to 3 times.

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