Vomitoxin degrading bacterium and application thereof

Abstract

The invention discloses a vomitoxin degrading bacterium and application thereof, and belongs to the technical field of microorganisms, the degrading bacterium is enterococcus faecium Z1, the strain is preserved in Guangdong Microbial Culture Collection Center on December 13, 2024, and the preservation number of the strain is GDMCC NO: 65569. The enterococcus faecium Z1 disclosed by the invention can be used for efficiently degrading the vomitoxin and obviously reducing the content of the vomitoxin in grains or feeds, and has a relatively good application value in the aspect of biological detoxification treatment of grains and feeds such as corn and wheat polluted by the vomitoxin.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of vomitoxin-degrading bacteria and applications thereof. Background Art

[0002] Deoxynivalenol (DON) is widely found in grains and feed materials such as barley, wheat, corn and oats. When humans and animals ingest food contaminated with DON, they will suffer from acute poisoning symptoms such as anorexia, vomiting, diarrhea, fever, unsteady standing and slow reaction. In severe cases, it will damage the hematopoietic system and cause death.

[0003] Humans and animals may experience a wide range of toxic effects after accidentally ingesting grains and food contaminated with vomitoxin. Low doses of vomitoxin can cause loss of appetite, slow growth, or even refusal to eat and stimulate immune responses, while high doses of vomitoxin can cause acute gastroenteritis accompanied by vomiting effects and can cause immunosuppression. After animals eat moldy feed, toxins and their metabolites remain in livestock products and their processed products, enter the human body through the food chain, and seriously threaten human health. Therefore, effectively controlling and eliminating the contamination of grains and feeds by mycotoxins is of great significance to improving animal production performance and ensuring human food safety.

[0004] Deoxynivalenol is such a serious hazard to animal husbandry and humans. Therefore, since the discovery of deoxynivalenol in the last century, scientists have been continuously studying the prevention, detoxification and detoxification methods of this type of toxin. So far, the known methods for removing deoxynivalenol from feed raw materials include physical, chemical and biological methods. In practical applications, physical and chemical detoxification methods are not applicable to the current feed industry and breeding industry. Because of their difficult operation, large quantities of feed and raw materials cannot be processed with such methods, and chemical detoxification treatment often reduces the nutritional quality and palatability of the feed, there is currently no effective method to prevent feed mycotoxin contamination. Microorganisms or biological enzymes have become a hot topic of concern due to the accuracy, effectiveness and environmental protection of their reactions.

[0005] In recent years, researchers have screened out microorganisms that can degrade DON from nature, but there have been no reports on Enterococcus faecalis degrading vomitoxin in feed. Summary of the invention

[0006] The technical problem to be solved by the present invention is to screen out new strains capable of degrading vomitoxin from a large number of natural samples, thereby solving the problem of vomitoxin contamination in feed and food causing harm to animal and human health.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is: a strain of vomitoxin-degrading bacteria, which is Enterococcus faecium Z1. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 13, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and its preservation number is GDMCC NO: 65569.

[0008] The present invention also provides a method for culturing vomitoxin-degrading bacteria: inoculating strain Enterococcus faecium.Z1 into liquid MRS culture medium and culturing at 37°C for 16 to 24 hours. 7 ~10 8 cfu / mL.

[0009] The present invention also provides an application of Enterococcus faecium Z1 in biological detoxification of vomitoxin.

[0010] The present invention also provides a vomitoxin-degrading bacterial agent, the active ingredient of which is the above-mentioned Enterococcus faecium Z1 or its fermentation product.

[0011] Preferably, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0012] The present invention also provides a method for degrading vomitoxin: using the above-mentioned bacteriolytic agent to treat materials containing vomitoxin, the materials including feed and grain and oil.

[0013] The present invention has the following beneficial effects: the Enterococcus faecium Z1 of the present invention can efficiently degrade vomitoxin, significantly reduce the content of vomitoxin in grains or feeds, and has good application value in the biological detoxification treatment of corn, wheat and other grains and feeds contaminated with vomitoxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a phylogenetic tree of the Enterococcus faecium 16S rDNA gene sequence of the present invention;

[0015] Figure 2 The colony morphology of Enterococcus faecium of the present invention is shown in FIG.

[0016] Figure 3 This is a colony morphology diagram of Enterococcus faecium after Gram staining of the present invention;

[0017] Figure 4 is the DNA electrophoresis diagram of Enterococcus faecium of the present invention;

[0018] Figure 5This is a diagram showing the 16S-PCR amplification results of Enterococcus faecium of the present invention;

[0019] Figure 6 is the growth curve of Enterococcus faecium of the present invention (Enterococcus faecium Z1 growth curve);

[0020] Figure 7 The results of the acid and alkali resistance test of Enterococcus faecium of the present invention are as follows;

[0021] Figure 8 This is a diagram showing the degradation effect of the DON standard product of Enterococcus faecium of the present invention;

[0022] Fig. 9 This is a diagram showing the effect of Enterococcus faecium in the present invention on degrading vomitoxin in WDGS. DETAILED DESCRIPTION

[0023] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with embodiments, but the content of the present invention is not limited to the following embodiments.

[0024] Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available or available to those skilled in the art; unless otherwise specified, the methods used in the examples of the present invention are all methods known to those skilled in the art.

[0025] <DON-degrading bacteria>

[0026] The degrading bacteria is Enterococcus faecium Z1, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 13, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and its collection number is GDMCC NO: 65569.

[0027] <Strain Screening>

[0028] 1) Sample processing

[0029] Weigh 1 g of soil from Baoqing County, Shuangyashan City, Heilongjiang Province into a sterile conical flask, add 10 mL of sterile water to each sample, mix thoroughly to obtain a suspension, and set aside.

[0030] 2) Initial screening

[0031] In a sterile environment, use a pipette to take 100 μL of the sample suspension after the above treatment and drop it onto LB, NA and MRS solid culture media containing DON (1 μg / mL), spread the suspension evenly, and then place the culture dish in a 37°C constant temperature incubator (aerobic and anaerobic environments) for 24-48 hours. According to the single colonies of different forms, pick the colonies with an inoculation loop, separate and purify them, preserve the bacteria in 20% glycerol, and number them in sequence according to the sample source, culture medium type and order of picking bacteria, and store them in a -20°C refrigerator for use.

[0032] 3) Rescreening

[0033] The purified primary screening strains were activated and then inoculated into MRS, NA and LB liquid culture media containing DON (100 μg / mL). Anaerobic bacteria were placed in a 37°C incubator for 48 hours. Aerobic bacteria were cultured at 37°C and 180 r / min for 48 hours. At the same time, MRS, NA and LB liquid culture media containing DON (100 μg / mL) were used as blank controls to screen out strains that efficiently degrade vomitoxin.

[0034] Specific dosage:

[0035] Control: 0.1 mL 100 ppm DON standard solution + 9.9 mL MRS culture medium.

[0036] Inoculation amount 5%: 0.1mL 100ppm DON standard solution + 0.5mL bacterial solution + 9.4mL MRS culture medium.

[0037] After adding the above proportions to the centrifuge tube, seal it with a sealing film and shake it in a shaker at 37°C for 24h (48h).

[0038] <Identification of strains>

[0039] Streak strain (Enterococcus faecium) Z1 onto the fermentation medium and observe the colony morphology (e.g. Figure 2 As shown), pick the colony for Gram staining, and observe the Gram-stained bacterial smear under the oil lens of an optical microscope. The bacteria are purple after Gram staining (as shown Figure 3 As shown), it turned out to be a Gram-positive (G+) bacterium.

[0040] 16S rDNA identification of strain Z1: Use kit to extract strain DNA, send the extracted DNA to test sequence, DNA electrophoresis and PCR amplification (such as Figure 4 and 5The PCR product is 1.2 kb. Sequence splicing and shearing were performed using SeqMan, and the results were input into the NCBI website for homology comparison. At the same time, homologous sequences were downloaded and imported into MEGA 13 software to finally construct a phylogenetic tree (as shown in Figure 1 The strain was identified as Enterococcus faecium.

[0041] The nucleotide sequence of the 16S rDNA gene of (Enterococcus faecium) Z1 is shown in SEQ ID NO:1.

[0042] <Determination of Growth Curve>

[0043] Scrape a loopful of the preserved slant culture medium and inoculate it into MRS liquid culture medium, culture it at 37°C, 180r / min for 12h, inoculate the above cultured bacterial solution into MRS liquid culture medium at a 1% inoculation rate for 12h to prepare a seed fermentation liquid. Transfer the cultured bacterial solution to MRS culture medium at a 1% inoculation rate, shake and mix well, place it in a constant temperature oscillator at 37°C, 180r / min and culture it for 36h. Take out the experimental group culture medium as a blank control every 2h to measure the OD600 nm value. Draw a growth curve (such as) with the culture time as the horizontal axis and the OD600 nm value as the vertical axis. Figure 6 shown).

[0044] <Acid and alkali resistance test>

[0045] Use 1 mol / L NaOH and 1 mol / L HCl solution to adjust the pH value of 100 mL of liquid LB medium to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, respectively, and add

[0046] 1 mL of seed fermentation liquid was cultured in a constant temperature oscillator at 37°C and 180 r / min for 24 h.

[0047] Use an ultraviolet spectrophotometer to measure the absorbance at 600 nm, set it to zero with a culture medium without bacteria and adjusted to pH, and draw a curve with pH as the horizontal axis and OD600 nm as the vertical axis (such as Figure 7 shown).

[0048] As can be seen from the figure, the effect of different pH on bacterial growth is shown in the figure. It can be seen from the figure that the bacteria grows more vigorously when the pH is 8-10, and the optimal pH is 10. The growth is hindered when the pH is 2-5 and 13-14.

[0049] <Determination of DON degradation rate of strains>

[0050] The determination of DON degradation rate was carried out according to the instructions of the DON enzyme-linked immunosorbent assay kit. The steps are as follows:

[0051] (1) After 48 hours of culture, the samples and the control group were diluted 5 times with deionized water and mixed thoroughly. After mixing, the samples were placed on an oscillator and shaken for 10 minutes.

[0052] (2) After thorough shaking, the sample was centrifuged at 4000 r / min for 5 min. After centrifugation, 10 mL of the supernatant was taken and added to 40 mL of deionized water. The mixture was thoroughly mixed by hand shaking for 5 s, and 50 μL was taken with a pipette for the next step.

[0053] (3) Open the reagent that has returned to room temperature, take out the required enzyme-labeled wells, and put the remaining enzyme-labeled wells back into the tin foil bag and seal it together with the desiccant for storage.

[0054] (4) The removed ELISA wells are numbered one by one corresponding to the standard and sample, and the locations of the standard and sample are recorded in detail.

[0055] (5) Use a pipette to draw 50 μL of standard and sample respectively and add them to the labeled wells, and then add 50 μL of DON anti-reagent to each well. After adding, gently shake to mix, and react at room temperature in the dark for 30 minutes.

[0056] (6) After the reaction, wash the wells 5 times with 250 μL / well of washing solution, with an interval of 5 seconds between each wash. After washing, add 50 μL / well of substrate solution A to each well, and then add 50 μL / well of substrate solution B. After adding, gently shake to mix, and react at room temperature in the dark for 15 minutes.

[0057] (7) Add 50 μL / well of stop solution and gently shake to mix. After addition, place the plate in an ELISA reader at 450 nm to measure the OD value of each well.

[0058] The calculation formula of DON degradation rate is:

[0059] DON degradation rate = (DON content in control group - DON content in sample group) / DON content in control group × 100%.

[0060] The degradation rate of DON standard product of Z1 strain was significantly reduced as follows (such as Figure 8 shown):

[0061] <Experiment on the degradation of vomitoxin in WDGS (wet corn distillers grains) by strains>

[0062] The activated Z1 was inoculated into 700g WDGS without any treatment at a 1% inoculum amount, mixed, and then placed in a 2.5L transparent fermentation bag with a one-way air valve and fermented at 37℃ for 10 days. The results are shown in the following table: Fig. 9 shown):

[0063] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A vomitoxin-degrading bacterium, characterized in that: The degrading bacteria is Enterococcus faecium Z1, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 13, 2024, and its preservation number is GDMCC NO: 65569.

2. The method for culturing vomitoxin-degrading bacteria according to claim 1, characterized in that: The strain Enterococcusfaecium.Z1 was inoculated into MRS medium and cultured at 37°C for 16 to 24 hours.

3. Use of the degradation bacterium Enterococcus faecium Z1 according to claim 1 in biological detoxification of vomitoxin.

4. A vomitoxin-degrading bacterial agent, characterized in that: The active ingredient of the bacterial agent is the Enterococcus faecium Z1 or its fermentation product as described in claim 1.

5. The vomitoxin-degrading bacterial agent according to claim 4, characterized in that: The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

6. A method for degrading vomitoxin, characterized in that: The material containing vomitoxin is treated with the bacteriolytic agent according to claim 4 or 5, and the material includes feed and grain and oil.

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