A culture medium and its culture method for promoting the degradation of vomitoxin by Slackia bacteria

Abstract

This invention discloses a culture medium and its cultivation method that can promote the degradation of DON by Slackia bacteria. The culture medium is formulated based on WCA medium, taking into account the different DON degradation efficiencies of Slackia bacteria under different metal ion conditions. Combined with appropriate cultivation methods, the efficiency of DON degradation by Slackia bacteria can be improved. This culture medium and its cultivation method can be applied to the cultivation of Slackia bacteria, significantly increasing the DON metabolism rate of Slackia strains by 30%-60%, showing promising application prospects for controlling mycotoxin contamination.

Description

Technical Field

[0001] This invention relates to the field of mycotoxin degradation technology, and more specifically, to a culture medium and a method thereof for promoting the detoxification of DON by Slackia bacteria. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by *Fusarium graminearum* and *Fusarium moniliforme* fungi that infect grain crops under conditions of high temperature and humidity, heavy rainfall, and poor storage. Dopamine nitrate (DON) belongs to type B trichothecete mycotoxins and widely contaminates grains such as corn, wheat, and barley, as well as their products. While DON has relatively low toxicity, it is widespread, has a high detection rate, and exhibits acute and chronic toxic effects, including enterotoxicity, cytotoxicity, immunotoxicity, and neurotoxicity. In humans and animals, after ingesting DON-contaminated food and feed, dopamine receptors in the vomiting center of the brainstem interact with DON, leading to vomiting; therefore, DON is also known as vomiting toxin. The molecular formula of DON is C0.05. 15 H 20 O6, chemically named 3α,7a,1,5-trihydroxy-12,13-epoxytrichosporon-9-en-8-one, is readily soluble in water, unstable in methanol, and can be stored for extended periods in acetonitrile and ethyl acetate. Because the DON structure is stable under high pressure, high temperature, and acidic conditions, it is not easily degraded during food and feed processing.

[0003] Currently, DON detoxification methods can be broadly categorized into physical, chemical, and biological methods. While physical and chemical methods can effectively remove DON, they have certain drawbacks. For example, some adsorbents have poor specificity or weak adsorption capacity, and while adsorbing DON, they also adsorb trace nutrients in food and feed, leading to nutrient loss. Chemical detoxification requires relatively harsh reaction conditions and can easily leave behind some potentially toxic substances, causing secondary pollution to the product. Biological detoxification utilizes detoxifying bacteria and enzymes to convert DON into low-toxicity or non-toxic metabolites, thereby reducing the harm of DON to human and animal health. Biological detoxification methods have advantages such as high specificity, mild reaction conditions, high efficiency, and environmental friendliness, and have little impact on the sensory characteristics and palatability of food and feed, making it a promising DON detoxification method.

[0004] Previous studies have shown that Slackia bacteria D-W1 and D-G6 can de-epoxygenate DON to the low-toxicity product DOM-1, and this de-epoxygenation reaction occurs concurrently with the growth of Slackia bacteria. However, due to the relatively slow growth of Slackia bacteria, their DON metabolism process often takes 48-72 hours to complete, which limits their application in practical production. Therefore, there is an urgent need for a culture medium and cultivation method to improve the efficiency of the DON de-epoxygenation reaction in Slackia bacteria, providing a foundation for their application in food or feed detoxification. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide a culture medium and a method for promoting the degradation of vomitoxin by Slackia bacteria.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention provides a culture medium that can promote the degradation of vomitoxin by Slackia bacteria, and the preparation steps are as follows:

[0008] Liquid culture medium: 1.0g glucose, 10.0g tryptone, 1.0g L-arginine, 0.0005g vitamin K1, 5.0g sodium chloride, 10.0g peptone, 0.005g heme chloride, 5.0g yeast extract, 1.0g sodium pyruvate, and supplements are mixed, dissolved in an appropriate amount of distilled water, and brought to a final volume of 1L. The mixture is then autoclaved at 121℃ for 20 minutes before use.

[0009] Solid culture medium: Mix 1.0 g glucose, 10.0 g tryptone, 1.0 g L-arginine, 10.0005 g vitamin K, 5.0 g sodium chloride, 10.0 g peptone, 0.005 g heme chloride, 5.0 g yeast extract, 1.0 g sodium pyruvate, supplement, and 15.0 g agar powder. Add an appropriate amount of distilled water to dissolve and bring the volume to 1 L. Autoclave at 121℃ for 20 min before use.

[0010] This invention also provides a culture method that can promote the degradation of vomitoxin by Slackia bacteria, comprising the following steps:

[0011] S1. Preparation of culture medium: Mix 1.0g glucose, 10.0g tryptone, 1.0g L-arginine, 0.0005g vitamin K1, 5.0g sodium chloride, 10.0g peptone, 0.005g heme chloride, 5.0g yeast extract, 1.0g sodium pyruvate, and supplements. Add an appropriate amount of distilled water to dissolve and bring the volume to 1L. Autoclave at 121℃ for 20 minutes and set aside.

[0012] S2, Inoculation: Inoculate the Slackia genus bacterial seed culture into the culture medium obtained in S1 and incubate statically.

[0013] S3. Incubation: Inoculate the culture obtained in S2 into fresh culture medium obtained in S1, and add DON or DON-containing substances for incubation and metabolism.

[0014] In a preferred embodiment, the supplement in the culture medium is composed of a mixture of 0.555 g of anhydrous calcium chloride, 0.634 g of anhydrous ferrous chloride, and 0.476 g of anhydrous magnesium chloride, i.e., the Ca in the culture medium... 2+ Mg 2+ and Fe 2+ The final concentrations are 10 mM, 5 mM and 5 mM, and can also be replaced by other salts containing equal amounts of metal ions.

[0015] In a preferred embodiment, the pH value of the culture medium is 8.2-8.5.

[0016] In the preferred embodiment, the culture temperature in steps S2 and S3 is 37°C, and the gas environment is a mixed gas environment containing 80% N2, 10% H2 and 10% CO2.

[0017] In the preferred embodiment, the bacterial strain inoculated in step S2 is Slackia bacteria: D-W1 (GDMCC NO: 63251).

[0018] In the preferred embodiment, the concentration of Slackia bacteria after inoculation in step S3 is 10. 4 CFU / mL.

[0019] The present invention has the following beneficial effects:

[0020] Compared to the previous Wilkins-Chalgren anaerobic broth (WCA) culture medium used for Slackia bacteria, the culture medium and its cultivation method provided in this invention can degrade DON to DOM-1 more efficiently. In some embodiments of this invention, the Slackia bacterium D-W1 strain was used for research. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 9, 2023, with accession number GDMCC NO: 63251. D-W1 is known to possess DON-degrading activity, and its DON degradation process is identical to that of Slackia bacterium D-G6. This invention can be applied to the DON degradation of Slackia bacteria in the feed and food industries, and has good application value.

[0021] Experiments showed that the addition of different concentrations of metal ions had varying effects on the DON degradation efficiency of Slackia bacteria D-W1. The effect was more pronounced with the addition of 10 mM Ca. 2+ 5mM Mg2+ or 5mM Fe 2+ Subsequently, the DON degradation efficiency of Slackia bacteria D-W1 increased by approximately 50%, 30%, and 20%, respectively. Attached Figure Description

[0022] Figure 1 The relationship between the growth and metabolism of DON in strain D-W1.

[0023] Figure 2 The effect of D-W1 on DON metabolism under divalent metal ion treatment. Detailed Implementation

[0024] Example 1: Growth and DON metabolic curves of Slackia bacteria D-W1

[0025] The Slackia bacterium D-W1 used in this invention was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 9, 2023, with accession number GDMCC NO: 63251. It was stored at -80℃ before use. The routine culture method for Slackia bacterium D-W1 is as follows:

[0026] S1. Prepare ordinary WCA medium: Mix 1.0 g glucose, 10.0 g casein peptone, 1.0 g L-arginine, 10.0 g vitamin K, 5.0 g sodium chloride, 10.0 g peptone, 0.005 g heme chloride, 5.0 g yeast extract, and 1.0 g sodium pyruvate. Add an appropriate amount of distilled water to dissolve and bring the volume to 1 L. Autoclave at 121℃ for 20 min and set aside.

[0027] S2. Inoculation: The seed culture of Slackia bacteria D-W1 was inoculated into the medium obtained in S1 and statically cultured at a temperature of 37°C, a pH of 8.0, and a gas environment containing a mixture of 80% N2, 10% H2 and 10% CO2.

[0028] S3. Incubation: The culture obtained in S2 was inoculated into fresh culture medium obtained in S1, and DON stock solution was added for incubation. Samples were collected every 12 hours to detect metabolic activity and observe bacterial growth. The samples were serially diluted to determine the amount of bacteria in each sample. After completion, statistical data were collected, and growth and DON metabolic curves of Slackia bacteria D-W1 were plotted.

[0029] The results are attached. Figure 1 As shown, the results indicate that the metabolism of DON by Slackia bacteria D-W1 is accompanied by its growth process.

[0030] Example 2Ca 2+ Mg 2+and Fe 2+ Promotes the deacyclooxygenation metabolism of DON in Slackia bacteria D-W1

[0031] The effects of different metal ions on the efficiency of Slackia bacteria in degrading vomitoxin were compared as follows:

[0032] S1. Preparation of WCA culture medium containing different concentrations of metal ions: The general preparation method is the same as S1 in Example 1, except that different concentrations of calcium chloride, magnesium chloride, manganese chloride, zinc chloride, copper chloride, or ferrous chloride are added, and a series of concentration gradients are set to adjust the Ca concentration in the culture medium. 2+ Mg 2+ Mn 2+ Zn 2+ Cu 2+ or Fe 2+ The concentrations were 5 mM, 10 mM, 15 mM, and 20 mM, respectively.

[0033] S2. Incubation: The cultures from step S2 of Example 1 were inoculated into the culture medium obtained in S1, and DON stock solution was added for incubation. After a period of time, samples were collected to detect DON metabolism.

[0034] The results are attached. Figure 2 As shown, the results indicate that Ca 2+ Mg 2+ and Fe 2+ At certain concentrations, all of them had the ability to promote the de-epoxy metabolism of DON by Slackia bacteria D-W1. Among them, Ca 2+ The 10 mM concentration showed the best promoting effect, with a DON degradation rate increased by approximately 50% compared to the control group.

Claims

1. A culture medium that promotes the degradation of DON by Slackia bacteria D-W1, characterized in that, This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 9, 2023, with the accession number GDMCC NO: 63251. The culture medium consists of glucose, tryptone, L-arginine, vitamin K1, sodium chloride, peptone, heme chloride, yeast extract, sodium pyruvate, and a supplement. The supplement is a mixture of 10 mM anhydrous calcium chloride, 5 mM anhydrous ferrous chloride, and 5 mM anhydrous magnesium chloride.

2. A method for preparing the culture medium according to claim 1, characterized in that, It is prepared by mixing glucose, tryptone, L-arginine, vitamin K1, sodium chloride, peptone, heme chloride, yeast extract, sodium pyruvate, and supplements.

3. A method for culturing using the culture medium of claim 1, characterized in that, Includes the following steps: S1 Culture Medium Preparation: Dissolve 1.0g glucose, 10.0g tryptone, 1.0g L-arginine, 0.0005g vitamin K1, 5.0g sodium chloride, 10.0g peptone, 0.005g heme chloride, 5.0g yeast extract, 1.0g sodium pyruvate, 1.11g anhydrous calcium chloride, 0.634g anhydrous ferrous chloride, and 0.476g anhydrous magnesium chloride in an appropriate amount of distilled water, and bring the volume to 1L. Autoclave at 121℃ for 20 minutes and set aside for use. S2 inoculation: The seed culture of Slackia bacteria D-W1 was inoculated into the culture medium obtained in S1 and then incubated statically. S3 incubation: The culture obtained from S2 is inoculated into fresh culture medium obtained from S1, and DON or DON-containing substances are added for incubation and metabolism.

4. The cultivation method according to claim 3, characterized in that, The pH value of the culture medium is 8.2-8.

5.

5. The cultivation method according to claim 3, characterized in that, In step S2, the seed culture of Slackia bacteria D-W1 is inoculated into culture medium S1 and statically cultured at a temperature of 37°C in a mixed gas environment containing 80% N2, 10% H2 and 10% CO2.

6. The cultivation method according to claim 3, characterized in that, Used to culture the Slackia bacteria D-W1 and improve its efficiency in metabolizing vomitoxin.

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