Probiotic bacillus subtilis capable of degrading various mycotoxins
By screening and isolating Bacillus subtilis H-1, ZEN's contamination problem in feed and food was solved, efficient degradation and nutrient utilization were achieved, and animal health and food safety were improved.
Patent Information
- Application Number
- CN202510445187.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-08-05
AI Technical Summary
In the prior art, zearalenone (ZEN) is widely present in feed and food, leading to threats to animals and human health. Traditional detoxification methods have problems with nutrient loss and environmental pollution. There are no efficient strains of microbial degradation methods.
A Bacillus subtilis H-1 was screened and isolated. By optimizing the culture conditions, ZEN was significantly degraded, animal health was improved, amylase and cellulase were produced, and nutrient utilization was improved.
Bacillus subtilis H-1 effectively degrades ZEN under optimized conditions, improves feed and food safety, improves animal health, reduces feed costs, and enhances immune function and reproductive health.
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Figure CN120424802A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of Bacillus subtilis, and specifically refers to a probiotic Bacillus subtilis that can degrade multiple mycotoxins. Background Art
[0002] Zearalenone (ZEN) is a mycotoxin widely present in various grains and feeds such as corn and barley. It can enter the human body and animals through the food chain, posing a threat to the health of humans and animals. ZEN mainly affects the liver, intestine, and immune system of animals. At the same time, by competing with 17β-estradiol for estrogen receptor binding, it can induce reproductive and fertility disorders in livestock and poultry. In addition, the residues of ZEN and its metabolites in animal products also indirectly endanger human health. In the investigation of the mycotoxin contamination situation of feed raw materials in China, it was found that the contamination rate of ZEN in feed raw materials was as high as 82.2%, indicating that the current contamination situation of ZEN in feed in China is not optimistic. At present, the detoxification methods of ZEN have attracted more and more attention. Traditional detoxification methods include physical detoxification and chemical detoxification, both of which have certain limitations, resulting in the loss of nutrients and the partial decomposition of some essential amino acids. The microbial degradation method has the advantages of high degradation efficiency, low environmental harm, and little reduction in the quality of feed products, and has quickly become a research hotspot at home and abroad. The strains used for degradation are mostly probiotics such as lactic acid bacteria, yeasts, and Bacillus.
[0003] Bacillus is famous for its heat-resistant, drought-resistant, radiation-resistant spores and its probiotic properties in the animal intestine. Among them, Bacillus subtilis is one of the microbial strains that can be used for feed grade announced by the Ministry of Agriculture and Rural Affairs of China. Research has found that Bacillus subtilis can inhibit the growth of pathogenic bacteria, promote the proliferation of beneficial bacteria, and maintain the balance of the intestinal microecosystem. In addition, Bacillus subtilis can also improve the intestinal environment through its metabolites, increase the digestion and absorption rate of nutrients, and activate the immune response of animals, enhancing the resistance to pathogens. Based on this, the purpose of this study is to isolate and screen Bacillus strains with the ability to degrade ZEN and good probiotic properties from soil samples, and evaluate their ability to improve the symptoms of ZEN poisoning in mice, providing a reference basis for their future use as feed additives and mycotoxin detoxifying agents. Summary of the Invention
[0004] The objective of the present invention is to provide a probiotic Bacillus subtilis strain capable of degrading multiple mycotoxins. The Bacillus subtilis H-1 of the present invention exhibits excellent degradation effects on zearalenone. Under optimized culture conditions, it can effectively reduce the content of ZEN in feed and food, reducing the potential threat of mycotoxins to animal and human health from the source, greatly improving the safety of agricultural products and feed, and ensuring the safe transmission of the food chain. The Bacillus subtilis H-1 of the present invention significantly promotes animal health, can improve the atrophy of the spleen and thymus, enhance the function of immune organs, and can significantly reduce the uterine and ovarian coefficients, alleviating the damage of ZEN to the female reproductive system and ensuring the reproductive health of animals. The strain H-1 can produce amylase and cellulase, and has good enzyme-producing ability. These enzymes can participate in the decomposition and digestion of nutrients in the animal intestine, helping to improve the utilization rate of carbohydrates in feed, promoting the absorption of nutrients by animals, reducing feed costs, and improving breeding efficiency.
[0005] To achieve the above objective, the technical solution adopted by the present invention is as follows: A probiotic Bacillus subtilis strain capable of degrading multiple mycotoxins. The isolated Bacillus subtilis H-1 has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M2024944 and the deposit date of May 15, 2024.
[0006] Furthermore, the similarity between the Bacillus subtilis H-1 and Bacillus subtilis DSM10 is 99.86%.
[0007] The present invention also provides a preparation method for a probiotic Bacillus subtilis strain capable of degrading multiple mycotoxins. The preparation method includes the following steps:
[0008] (1) Isolation and screening of the strain: Weigh 1 g of soil sample, place it in 50 mL of broth medium, treat it in a water bath at 80 °C for 15 min, and then incubate it at 37 °C with constant shaking for 24 h. Take 1 mL of the cultured bacterial solution, perform gradient dilution with sterile physiological saline, and pipette 10 -3 、10 -4 、10 -5 100 μL of each gradient treatment solution is spread on the plate and incubated at 37 °C for 24 h. Single colonies are picked according to colony morphology for microscopic examination, and spore-producing bacilli are selected for streak isolation and purification. The purified bacilli are stored at 4 °C in the refrigerator.
[0009] (2) Determination of the ZEN degradation rate of each strain: After activating and culturing each strain, inoculate it into a broth medium and culture at 37 °C for 24 h. After the culture is completed, take 900 μL of the fermentation broth of each strain and add 100 μL of the ZEN standard product dilution solution into a 1.5 mL sterile centrifuge tube as the experimental group. At the same time, take 900 μL of the broth medium and add 100 μL of the ZEN standard product dilution solution as a blank control. After culturing the experimental group and the control group at 37 °C for 72 h, detect the ZEN degradation rate of each strain, and select the strain with the highest degradation rate for subsequent experiments;
[0010] (3) Physiological and biochemical identification of the strain: Conduct sugar fermentation tests, V-P tests, and gelatin liquefaction tests on the strain for preliminary identification;
[0011] (4) Molecular identification: Amplify the 16S rDNA gene fragment of the strain. The primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd. Detect and recover the target product by agarose gel electrophoresis, send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, perform BLAST alignment of the sequencing results in NCBI, and construct a phylogenetic evolution tree using MEGA 11.0 software.
[0012] Furthermore, the soil sample in step (1) is from the rhizosphere soil of peony roots in Yuzhong Campus.
[0013] Furthermore, the formula for the ZEN degradation rate of the strain in step (2) is: Strain mycotoxin degradation rate = (1 - experimental group toxin concentration / control group toxin concentration) × 100%.
[0014] Furthermore, the universal primers amplified in step (4) are: 27F 5′-AGAGTTTGA-TCCTGGCTCAG-3′ and 1492R 5′-GGTTACCTTGTTA-CGACTT-3′.
[0015] Furthermore, the amplification system in step (4) is: Premix Taq 12.5 μL, 1 μL each of the upstream and downstream primers, 1 μL of genomic DNA, 9.5 μL of ddH2O.
[0016] Furthermore, the PCR amplification program in step (4): Pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 60 s, a total of 30 cycles; extension at 72 °C for another 10 min; preservation at 4 °C.
[0017] The beneficial effects achieved by the present invention using the above structure are as follows: (1) Bacillus subtilis H-1 of the present invention exhibits excellent degradation effect on zearalenone. Under optimized culture conditions, it can effectively reduce the content of ZEN in feed and food, reduce the potential threat of mycotoxins to animal and human health from the source, greatly improve the safety of agricultural products and feed, and ensure the safe transmission of the food chain; (2) Bacillus subtilis H-1 of the present invention has a significant promoting effect on animal health, can improve the atrophy of the spleen and thymus, enhance the function of immune organs, and can significantly reduce the uterine and ovarian coefficients, relieve the damage of ZEN to the female reproductive system, and ensure the reproductive health of animals; (3) The strain H-1 of the present invention can produce amylase and cellulase, and has good enzyme production ability. These enzymes can participate in the decomposition and digestion of nutrients in the animal intestine, help improve the utilization rate of carbohydrates in feed, promote the absorption of nutrients by animals, reduce feed costs, and improve breeding efficiency. Brief Description of the Drawings
[0018] Figure 1 ZEN degradation rate of each strain;
[0019] Figure 2 Identification result of the 16S rDNA gene sequence of the isolated strain;
[0020] Figure 3 Phylogenetic tree of the 16S rDNA sequence of the isolated strain;
[0021] Figure 4 Effect of H-1 on the body weight of ZEN-poisoned mice;
[0022] Figure 5 Enzyme production of the strain.
[0023] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention. Detailed Embodiments
[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0025] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only and do not limit the content of this application.
[0026] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods; in the test materials and test strains used in the following examples, unless otherwise specified, they are all obtained by purchasing from commercial channels.
[0027] Example 1
[0028] Isolation and screening of strains
[0029] Weigh 1 g of the rhizosphere soil sample of Paeonia suffruticosa from Yuzhong Campus, place it in 50 mL of broth medium, treat it in a water bath at 80 °C for 15 min, then incubate it at 37 °C with constant shaking for 24 h. Take 1 mL of the cultured bacterial liquid, perform gradient dilution with sterile normal saline, and pipette 100 μL of the treatment liquids of each gradient of 10 -3 、10 -4 、10 -5 onto the plate, incubate it at 37 °C for 24 h, pick single colonies for microscopic examination according to the colony morphology, select the spore-forming bacteria for streak isolation and purification, and store the purified spore-forming bacteria at 4 °C.
[0030] Example 2
[0031] Determination of the ZEN degradation rate of each strain
[0032] After activating and culturing each strain, inoculate it into the broth medium and culture it at 37 °C for 24 h. After the culture is completed, take 900 μL of the fermentation broth of each strain and add 100 μL of the ZEN standard product dilution solution into a 1.5 mL sterile centrifuge tube as the experimental group. At the same time, take 900 μL of the broth medium and add 100 μL of the ZEN standard product dilution solution as the blank control. After culturing the experimental group and the control group at 37 °C for 72 h, detect the ZEN degradation rate of each strain, and select the strain with the highest degradation rate for subsequent tests. The ZEN degradation rate of the strain is calculated using the following formula: strain mycotoxin degradation rate = (1 - experimental group toxin concentration / control group toxin concentration) × 100%, and the results are as Figure 1 shown: The ZEN degradation rate of strain H-1 is the highest, reaching 78.3%, and it has a high ability to degrade zearalenone.
[0033] Example 3
[0034] Physiological and biochemical identification of strains
[0035] Sugar fermentation test, V-P test, gelatin liquefaction test, etc. were carried out on the strain. The strain was preliminarily identified by referring to "Bergey's Manual of Determinative Bacteriology" and "Manual of Systematic Identification of Common Bacteria". As shown in Table 1, the biochemical identification results of strain H-1 showed positive results in V-P, xylose, sucrose, arabinose, maltose, glucose, and gelatin liquefaction tests, and negative results in lactose, mannitol, and hydrogen sulfide tests. Comparing the test results with "Bergey's Manual of Determinative Bacteriology" and "Manual of Systematic Identification of Common Bacteria", it was preliminarily determined that the isolated strain belongs to the genus Bacillus.
[0036] Table 1 Biochemical Identification Results of Strains
[0037]
[0038] "+" indicates a positive reaction, and "-" indicates a negative reaction.
[0039] Example 4
[0040] Molecular Identification of Strains
[0041] The 16S rDNA gene fragment of the strain was amplified. The universal primers 27F 5′-AGAGTTTGA-TCCTGGCTCAG-3′ and 1492R 5′-GGTTACCTTGTTA-CGACTT-3′ were amplified, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Amplification system: Premix Taq 12.5 μL, 1 μL each of upstream and downstream primers, 1 μL of genomic DNA, and 9.5 μL of ddH2O. PCR amplification program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 60 s, for a total of 30 cycles; extension at 72°C for another 10 min; preservation at 4°C. The target product was detected by agarose gel electrophoresis and recovered, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared by BLAST in NCBI, and a phylogenetic evolution tree was constructed using MEGA11.0 software.
[0042] As Figure 2 shown, the fragment size of the 16S rDNA gene amplification product of the strain was about 1500 bp. The strain sequence was compared in the NCBI database, and a phylogenetic evolution tree was constructed using MEGA software ( Figure 3 ). The similarity between the isolated strain and Bacillius subtilis DSM10 was 99.86%. Combining the morphological characteristics and physiological and biochemical test results of the strain, it was determined that the strain was Bacillius subtilis and named Bacillius subtilis H-1.
[0043] Experimental Example 1
[0044] Effect of the strain on ZEN-intoxicated mice
[0045] Thirty mice were randomly divided into three groups. Mice in the control group were fed a basal diet and orally administered normal saline. Mice in the ZEN group were orally administered a diluted solution of ZEN standard (50 mg / kg BW) based on the basal diet. Mice in the H-1 group were orally administered a diluted solution of ZEN standard (50 mg / kg BW) and H-1 culture solution (1×108 CFU / mL) based on the basal diet. The experimental period was 14 days. During the experiment, the mice had free access to food and water, and the body weight changes, mental state, and growth status of the mice were observed and recorded. At the end of the experiment, blood was collected, serum was separated, and the concentrations of estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the serum were measured using an ELISA kit, and the activities of ALT and AST in the serum were also measured. The liver, spleen, thymus, uterus, and ovaries were collected and the organ coefficients were calculated. Organ coefficient = (organ weight / mouse body weight)×100%.
[0046] Result analysis: As Figure 4 shown, ZEN had a certain impact on the body weight of mice, making their weight gain slow. ZEN could significantly reduce the growth performance of mice (P<0.01), while Bacillus subtilis H-1 could alleviate the impact of ZEN on the body weight of mice and had a significant improvement effect on the growth performance affected by ZEN (P<0.01).
[0047] Experimental example 2
[0048] Tolerance test of artificial intestinal juice and artificial gastric juice
[0049] 100 μL of bacterial liquid was added to 900 μL of artificial intestinal juice (artificial gastric juice) and mixed evenly, and then cultured at 37°C and 200 rpm for 0, 1, 2, 3, 4, 5, 6 h. After the culture was completed, gradient dilution and plate coating were carried out, and then cultured at 37°C for 24 h. The plates with the number of colonies in the range of 30 - 300 were selected for counting, and each experiment was repeated 3 times. The viable bacteria count at 0 h was used as the control group, and the survival rate at different treatment times was calculated. Tolerance rate of artificial intestinal juice (artificial gastric juice) (%) = (number of colonies at different treatment times / number of colonies at 0 h)×100%. Result analysis: The survival rate of the isolated strain reached 45.5% after being treated in artificial gastric juice for 6 h, and reached 64.1% after being treated in artificial intestinal juice for 6 h, indicating that the strain had good tolerance to both artificial intestinal juice and artificial gastric juice and could grow and reproduce in the intestine and play its probiotic performance.
[0050] Experimental example 3
[0051] Enzyme production test
[0052] Under aseptic conditions, use an inoculating loop to pick up Bacillus subtilis and spot inoculate it on the cellulase selection medium, protease selection medium, and amylase selection medium respectively, and culture at 37 °C for 48 h. Observe directly the situation of the clear zone around the colonies on the protease selection medium. For the cellulase selection medium, stain it with 1 mg / mL Congo red, and then elute it with 1 mol / L NaCl solution until a clear zone appears. After adding iodine solution to the amylase selection medium for color development, observe the situation of the clear zone around the colonies.
[0053] Result analysis: As Figure 5 shown, strain H-1 can produce amylase and cellulase, and has good enzyme-producing ability. The strain does not produce protease (A: amylase; B: cellulase; C: protease).
[0054] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
[0055] The above describes the present invention and its embodiments. This description is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural modes and embodiments without creative efforts without departing from the purpose of the present invention, they should all fall within the protection scope of the present invention.
Claims
1. A probiotic strain of Bacillus subtilis capable of degrading multiple mycotoxins, characterized by: The isolated Bacillus subtilis H-1 was deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M2024944.
2. The probiotic Bacillus subtilis capable of degrading multiple mycotoxins according to claim 1, characterized in that: The similarity between the Bacillus subtilis H-1 and Bacillus subtilis DSM10 is 99.86%.
3. A method for preparing the probiotic Bacillus subtilis capable of degrading multiple mycotoxins according to claim 2, characterized in that: The preparation method comprises the following steps: (1) Isolation and screening of strains: Weigh 1 g of soil sample and place it in 50 mL of broth culture medium. Treat it in a water bath at 80°C for 15 min, and then culture it at 37°C with constant temperature shaking for 24 h. Take 1 mL of the culture solution and dilute it with sterile saline for gradient dilution. Take 10 -3 , 10 -4 , 10 -5 100 μL of each gradient treatment solution was spread on a plate and incubated at 37°C for 24 h. Single colonies were picked for microscopic examination based on their morphology. Spore-forming Bacillus was selected for streak isolation and purification. The purified Bacillus was refrigerated and stored at 4°C. (2) Determination of ZEN degradation rate of each strain: After activation and culture, each strain was inoculated into broth culture medium and cultured at 37°C for 24 h. After the culture was completed, 900 μL of the fermentation broth of each strain was added to 100 μL of ZEN standard dilution solution in a 1.5 mL sterile centrifuge tube as the experimental group. At the same time, 900 μL of broth culture medium was added to 100 μL of ZEN standard dilution solution as the blank control. After the experimental group and the control group were cultured at 37°C for 72 h, the ZEN degradation rate of each strain was measured. The strain with the highest degradation rate was selected for subsequent experiments. (3) Physiological and biochemical identification of the strain: sugar fermentation test, VP test and gelatin liquefaction test were performed on the strain to conduct preliminary identification of the strain; (4) Molecular identification: The 16S rDNA gene fragment of the strain was amplified. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The target product was detected and recovered by agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared by BLAST in NCBI, and the phylogenetic tree was constructed using MEGA11.0 software.
4. The probiotic Bacillus subtilis capable of degrading multiple mycotoxins according to claim 3, characterized in that: The soil sample in step (1) is from the peony rhizosphere soil of Yuzhong campus.
5. The probiotic Bacillus subtilis capable of degrading multiple mycotoxins according to claim 4, characterized in that: The formula for the degradation rate of the strain ZEN in step (2) is: mycotoxin degradation rate of the strain = (1-toxin concentration of the experimental group / toxin concentration of the control group) × 100%.
6. The probiotic Bacillus subtilis capable of degrading multiple mycotoxins according to claim 5, characterized in that: The universal primers for amplification in step (4) are: 27F 5′-AGAGTTTGA-TCCTGGCTCAG-3′ and 1492R 5′-GGTTACCTTGTTA-CGACTT-3′.
7. The probiotic Bacillus subtilis capable of degrading multiple mycotoxins according to claim 6, characterized in that: The amplification system in step (4) is: 12.5 μL of Premix Taq, 1 μL of upstream and downstream primers, 1 μL of genomic DNA, and 9.5 μL of ddH2O.
8. The probiotic Bacillus subtilis capable of degrading multiple mycotoxins according to claim 7, characterized in that: The PCR amplification procedure in step (4) is as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 60 s, for a total of 30 cycles; further extension at 72°C for 10 min; and storage at 4°C.