CN110804570A - Bacillus beijerinckii for simultaneously degrading zearalenone and aflatoxin and application thereof - Google Patents
Bacillus beijerinckii for simultaneously degrading zearalenone and aflatoxin and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of agricultural biology, in particular to Bacillus velezensis ANSB01E CGMCC No.17328 and application thereof. The Bacillus beilesensis can efficiently degrade zearalenone and aflatoxin at the same time, and the degradation rate of zearalenone and aflatoxin after reaction for 48 hours is over 90 percent. The bacillus beilesensis provided by the invention has the advantages of high toxin degrading efficiency, strong specificity and mild effect, does not destroy nutrient components in feed, and is suitable for biological detoxification of mycotoxin in grains, feed and food.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to Bacillus beijerinckii (Bacillus velezensis) ANSB01E for simultaneously degrading zearalenone and aflatoxin and application thereof.
Background
Mycotoxins are toxic secondary metabolites produced by fungi such as Aspergillus spp, Fusarium spp and Penicillium spp during growth. About 300 mycotoxins with toxic effects on humans and animals have been reported, among which Aflatoxin (Aflatoxin), Zearalenone (Zearalenone), trichothecene toxoid (Trichothecenes), Ochratoxin (Ochratoxin), and fumonisin (Fumonisins) pose the most serious threat to the animal industry. The traditional physical and chemical elimination method has the defects of unstable effect, large loss of nutrient components, influence on the palatability of the feed, difficulty in large-scale production and the like, and cannot be widely applied to actual production. The microorganism and the biological enzyme degradation have the characteristics and advantages of high detoxification efficiency, strong specificity, no pollution to feed and environment and the like, thereby being concerned by researchers. In recent years, microbial strains for degrading zearalenone and some research reports for degrading aflatoxin strains have been reported, most of the reports are that a single strain or a mixed strain degrades one toxin, the research on the simultaneous degradation of multiple toxins by a single strain is very little, and no research report on the simultaneous degradation of zearalenone and aflatoxin by bacillus beilisianus exists at present. The invention discloses bacillus beilaisi capable of degrading zearalenone and aflatoxin simultaneously, which has very important significance for effectively controlling and eliminating pollution of mycotoxin to grains and feeds, improving animal production performance and ensuring human food safety.
Disclosure of Invention
The invention aims to solve the technical problem of screening beneficial bacteria capable of degrading zearalenone and aflatoxin simultaneously from a large number of natural samples, thereby solving the problem of mycotoxin pollution in grains, feeds and foods.
In view of the above, the invention provides a Bacillus subtilis (Bacillus velezensis) for simultaneously degrading zearalenone and aflatoxin and application thereof in mycotoxin detoxification.
Another object of the present invention is to provide the use of Bacillus belgii as described above.
Another object of the present invention is to provide a mycotoxin biodegradation agent containing the Bacillus belgii.
Another object of the present invention is to provide a method for producing the above-mentioned mycotoxin biodegradation agent containing Bacillus belgii.
Another object of the present invention is to provide the use of the above-mentioned mycotoxin biodegradation agent containing Bacillus belgii.
In order to achieve the above object, the present invention provides the following technical solutions:
the Bacillus velezensis ANSB01E of the invention has been preserved in China general microbiological culture Collection center in 2019 at 13.03.13.A preservation address is the microbiological research institute of China academy of sciences No. 3, North West Lu No.1 Hotel, Chaoyang, Beijing, and the preservation number is CGMCC No. 17328.
The Bacillus beijerinckii ANSB01E capable of degrading zearalenone and aflatoxin simultaneously is obtained by separating from chicken intestinal tract meal through complex screening work.
The invention also provides application of the bacillus beilesensis, in particular application in mycotoxin detoxification, and the bacillus beilesensis can efficiently degrade mycotoxins such as zearalenone and aflatoxin.
The invention also provides a mycotoxin biodegradation agent, which comprises the bacillus beilesensis and a physiologically acceptable carrier, wherein the physiologically acceptable carrier comprises one or more of yeast cell culture, wheat bran, maltodextrin, cyclodextrin, rice bran, cane sugar, starch, talcum powder, montmorillonite and plant extract.
The mycotoxin biodegradability agent also includes a microecological preparation, which includes but is not limited to one or more of bacillus subtilis, bacillus licheniformis, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, lactobacillus acidophilus, lactobacillus casei, lactobacillus delbrueckii subsp lactis, lactobacillus plantarum, pediococcus acidilactici, pediococcus pentosaceus, candida utilis, bifidobacterium infantis, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus oryzae, bacillus lentus, bacillus pumilus, lactobacillus cellobiosus, lactobacillus fermentum or lactobacillus delbrueckii subsp bulgaricus, preferably, the mycotoxin biodegradation agent further contains at least one of propionibacterium, lactobacillus buchneri and lactobacillus paracasei when being used for silage or cattle feed; or the mycotoxin biodegradation agent also contains bacillus coagulans and/or brevibacillus laterosporus when being used for feeds of poultry, pigs and aquaculture animals.
The mycotoxin biodegrading agents described above may also include additional enzymes including, but not limited to, deoxynivalenol epoxide hydrolase, carboxypeptidase, aflatoxin oxidase, zearalenone lactonase, fumonisin carboxyl esterase, fumonisin aminotransferase, laccase, aminopolyol amine oxidase, aspergillus niger aspartic protease, elastase, aminopeptidase, pepsin-like protease, trypsin-like protease, bacterial protease, amylase, arabinase, arabinofuranosidase, catalase, cellulase, hemicellulase, lignin degrading enzyme, chitinase, rennin, cutinase, deoxyribonuclease, epimerase, esterase, galactosidase, glucanase, glucanolytic enzyme, endoglucanase, glucanolytic enzyme, glucoamylase, and other enzymes, Glucose oxidase, glucosidase, glucuronidase, hexose oxidase, hydrolase, invertase, isomerase, lipolytic enzyme, lyase, mannosidase, xylanase, oxidase, oxidoreductase, aldoketoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methylesterase, pectinolytic enzyme, peroxidase, manganese peroxidase, phenol oxidase, phytase, polygalacturonase, alcohol dehydrogenase, rhamnogalacturonase, ribonuclease, transferase, transporter, transglutaminase, epoxide hydrolase, hexose oxidase, acid phosphatase, and enzymes involved in starch metabolism, fiber degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism.
In the above mycotoxin biodegradation agent, the content of the microecologics/additional enzymes is 0-20%.
The preparation method of the mycotoxin biodegradation agent comprises the following steps: preparing a mixture containing Bacillus belgii ANSB01E bacterial cells and a physiologically acceptable carrier, and further mixing the mixture with a microecological preparation or another enzyme according to a certain proportion to obtain a mycotoxin biodegradation agent; the content of Bacillus beleisi ANSB01E cells in the mycotoxin biodegradation agent is 1-10%.
Among the above additives, bacillus belgii ANSB01E bacterial cells: physiologically acceptable carrier: the mass ratio of the probiotics/additional enzymes may be: 1: 79: 20 or 2: 78: 20 or 3: 77: 20 or 4: 76: 20 or 5: 75: 20 or 6: 74: 20 or 7: 73: 20 or 8: 72: 20 or 9: 71: 20 or 10: 70: 20.
The invention also provides a method and application of the mycotoxin biodegradation agent in mycotoxin detoxification.
The method and use for degrading mycotoxins, characterized in that a material containing mycotoxins is treated with the Bacillus belgii or the mycotoxin biodegrading agent; the material includes, but is not limited to, one or more of food, feed and raw materials thereof, grain and by-products of processing thereof, grain oil, milk, aged grain, tea, fruits, fruit juices, or chinese herbal medicines.
Biological preservation Instructions
ANSB01E, class name: bacillus velezensis, was deposited in China general microbiological culture Collection center at 13.03.2019, with the address of the institute of microbiology, China academy of sciences, institute of microbiology, No. 3, Siro 1, North Chen, south Kogyo, Beijing, and the accession number of CGMCC No. 17328.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the cell morphology under electron microscope of strain ANSB01E of the present invention;
FIG. 2 shows a liquid chromatogram of the degradation of zearalenone by strain ANSB01E of the invention;
FIG. 3 shows a liquid chromatogram of the degradation of aflatoxin B1 by strain ANSB01E of the invention;
figure 4 shows the effect of strain ANSB01E of the present invention on the degradation of ZEN in ZEN moldy corn.
Detailed Description
The invention discloses Bacillus belgii ANSB01E and application thereof in mycotoxin detoxification, and a person skilled in the art can appropriately improve process parameters for realization by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention is further illustrated by the following examples:
example 1 screening of strains that degrade zearalenone and aflatoxin simultaneously
29 samples of soil, mildewed feed, pig and chicken intestinal tract cachexia and the like are collected and used for screening microorganisms for simultaneously degrading zearalenone and aflatoxin. 1g of the sample was dissolved in sterile PBS buffer, spread on LB plates, and cultured at 37 ℃ for 24 hours. 20 single colonies were randomly picked from each plate and streaked on LB plates 3 times to obtain purified strains. After the isolated and purified strains were cultured in LB medium at 37 ℃ for 24 hours, the degradation rate of each strain to ZEN and AFB1 was examined.
The reaction conditions are as follows: 990 μ L of strain fermentation broth was incubated with 10 μ L of ZEN (200 μ g/mL) at 37 ℃ for 48h, 990 μ L of sterile PBS was used as a control with 10 μ L of ZEN (200 μ g/mL), after the reaction was completed, the reaction was terminated by adding an equal volume of methanol, the concentration of ZEN was measured by HPLC, and the degradation rate was calculated.
Test results show that one of the strains ANSB01E (shown in figure 1 in an electron microscope morphology) derived from the cecal carcasses of the broiler chickens has higher degradation effect on ZEN, and the degradation rate is 95% (shown in figure 2).
And then incubating the strain fermentation liquor with AFB1 to verify whether the strain fermentation liquor can degrade aflatoxin at the same time. The reaction conditions are as follows: 990 mu L of strain fermentation liquor and 10 mu L of AFB1(10 mu g/mL) are incubated for 48h at 37 ℃, 990 mu L of sterile PBS and 10 mu L of AFB1(10 mu g/mL) are used as controls, equal volume of methanol is added to stop the reaction after the reaction is finished, the concentration of AFB1 is detected by HPLC, and the degradation rate is calculated.
The test results show that ANSB01E can degrade AFB1, and the degradation rate of the AFB1 is 90% (as shown in FIG. 3).
Example 2 degrading Effect of Bacillus belgii ANSB01E on ZEN in naturally mouldy maize
Firstly, smashing Zea mays with ZEN mildewed, weighing 10g of the smashed Zea mays, re-suspending the weighed Zea mays in 40mL of distilled water, sterilizing the weighed Zea mays for 15min at 121 ℃, detecting the ZEN concentration in a corn meal leaching solution to be 0.74 mu g/mL, respectively inoculating 10% of Bacillus belgii ANSB01E or LB culture medium in the corn meal leaching solution, incubating the obtained product at 37 ℃ for 48h, respectively, then sampling the obtained product after incubating the obtained product for 0, 12, 24 and 48h, and detecting the ZEN content by HPLC.
The results are shown in FIG. 4, and the degradation rate of ZEN in naturally moldy corn by Bacillus belgii ANSB01E is 73%.
Example 3 degradation of Bacillus belgii ANSB01E on zearalenol, zearalanol, AFM1 and AFG1
Zearalenol, zearalanol, AFM1 and AFG1 were dissolved in dimethyl sulfoxide respectively and tested according to the following reaction system: 990 mu L of Bacillus beilaisi fermentation liquor and 10 mu L of toxin, wherein the concentration of zearalenol or zearalenol in the system is 10 mu g/mL, and the concentration of AFM1 or AFG1 is 1 mu g/mL; PBS was used as a control in the toxin reaction; after reacting for 48h at 37 ℃, 1mL of methanol is added to stop the reaction, and the content of each toxin is detected by adopting high performance liquid chromatography. The efficiency of bacillus belezii in degrading zearalenol, zearalanol, AFM1 and AFG1 is shown in table 1.
TABLE 1 efficiency of Bacillus belgii for degrading zearalenol, zearalanol, AFM1 and AFG1
Example 4A mycotoxin biodegradation agent and a method for preparing the same
The mycotoxin biodegradation agent is obtained by weighing 90% of the carrier (wheat bran and starch are mixed according to the mass ratio of 2: 1) of the total mass of the additive, and then mixing the carrier with the bacillus beilesensis ANSB01E of example 1 according to the proportion of 10% of the total mass of the additive.
Example 5A mycotoxin biodegradation agent and a method for preparing the same
Weighing a carrier accounting for 9% of the total mass of the mycotoxin biodegradation agent (mixing sucrose and starch according to the mass ratio of 1: 1), then mixing the carrier into the Bacillus belezii ANSB01E in example 1 according to the mass ratio of 1% of the total mass of the additive, and finally mixing the mixed preparation powder of the Bacillus licheniformis and the Bacillus amyloliquefaciens according to 90% of the total mass of the additive (the mass ratio of the Bacillus licheniformis to the Bacillus amyloliquefaciens is 2: 1) to obtain the mycotoxin biodegradation agent.
Example 6A mycotoxin biodegradation agent and a method for preparing the same
The mycotoxin biodegradation agent is obtained by firstly mixing rice bran accounting for 70% of the total mass of the additive with lactobacillus plantarum accounting for 10% of the total mass of the additive, then mixing the rice bran and lactobacillus plantarum ANSB01E which is described in example 1 according to the proportion of 10% of the total mass of the additive, and finally mixing the rice bran and lactobacillus plantarum ANSB01E according to the proportion of 10% of the total mass of the additive.
Example 7A mycotoxin biodegradation agent and a method for preparing the same
Firstly, sucrose accounting for 70 percent of the total mass of the additive is mixed with bifidobacterium animalis accounting for 20 percent of the total mass of the additive, then the bifidobacterium beijerinckii ANSB01E in the embodiment 1 is mixed according to the proportion of 5 percent of the total mass of the additive, amylase is mixed according to the proportion of 5 percent of the total mass of the additive, and finally plant extract is mixed according to 1 percent of the total mass of the additive, so as to obtain the mycotoxin biodegradation agent.
Example 8 treatment of moldy wheat with ZEN with a mycotoxin biodegradation agent
Wheat treated with a mycotoxin biodegradation agent described in example 5 and containing ZEN (ZEN 4ppm) was mixed at a ratio of 0.1% and digested in vitro for 24h in simulated animal gastrointestinal fluids for degradation of ZEN in feed.
Simulated gastric fluid: 2g of wheat containing ZEN was weighed, 2mg of a degradation agent was added, and the mixture was put into a 100mL Erlenmeyer flask, and 25mL of PBS (0.1M pH6.0) was added to adjust the pH to 6.8, followed by mixing. Adding 1mL of prepared amylase solution, digesting for 2h at 39 ℃ and 150 r/min. 10mL of 0.2M HCl was added, the pH was adjusted to 2.0 with 1M HCl or 1M NaOH solution, 1mL of freshly prepared acidic protease (50000U/g) was added, mixed well, capped with parafilm, and shake-cultured at 39 ℃ for 6h (150 r/min).
Simulating small intestine liquid: after incubation with simulated gastric fluid for 6h, 5mL of 0.6M NaOH solution was added, the pH was adjusted to 6.8 with 1M HCl or 1M NaOH, and a freshly prepared suspension of the exogenous enzyme in the intestine (protease: amylase: lipase: 3: 1) was added, sealed with parafilm, and incubated in a constant temperature shaker at 39 ℃ for 18h (150r/min) each.
The degradation rate of ZEN after the end of the reaction was determined to be 89.63%.
Example 9 treatment of moldy peanut meal containing AFB1 with a mycotoxin biodegradation agent
The mycotoxin biodegradation agent of example 7 is used for treating mildewed peanut meal containing AFB1 (AFB1 ppb) and is mixed according to the proportion of 0.1 percent, and the mildewed peanut meal is digested in vitro in animal gastrointestinal fluids for 24 hours and is used for degrading AFB1 in feed.
Simulated gastric fluid: 2g of the moldy peanut meal containing AFB1 was weighed, 2mg of a degradation agent was added, the mixture was put into a 100mL Erlenmeyer flask, 25mL of PBS (0.1M pH6.0) was added, the pH was adjusted to 6.8, and the mixture was mixed well. Adding 1mL of prepared amylase solution, digesting for 2h at 39 ℃ and 150 r/min. Adding 10mL of 0.2M HCl, adjusting pH to 2.0 with 1M HCl or 1M NaOH solution, adding 1mL of freshly prepared acidic protease (50000U/g), mixing, sealing with parafilm, and shaking at 39 deg.C for 6h (150 r/min).
Simulating small intestine liquid: after incubation with simulated gastric fluid for 6h, 5mL of 0.6M NaOH solution was added, the pH was adjusted to 6.8 with 1M HCl or 1M NaOH, and a freshly prepared suspension of the exogenous enzyme in the intestine (protease: amylase: lipase: 3: 1) was added, sealed with parafilm, and incubated in a constant temperature shaker at 39 ℃ for 18h (150r/min) each.
After the reaction, the degradation rate of AFB1 was determined to be 83.63%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The Bacillus beilaisi ANSB01E for degrading zearalenone and aflatoxin simultaneously is deposited in China general microbiological culture Collection center with the preservation address of China academy of sciences microorganism institute of China No. 3, West Lu No.1, Beijing, Chaoyang, and the preservation number is CGMCC No. 17328.
2. A mycotoxin biodegradation agent comprising Bacillus belgii ANSB01E according to claim 1 in an amount of 1-10% and a physiologically acceptable carrier.
3. The mycotoxin biodegrading agent of claim 2, wherein the physiologically acceptable carrier comprises, but is not limited to, one or a mixture of two of yeast cell culture, wheat bran, maltodextrin, cyclodextrin, rice bran, sucrose, starch, talc, montmorillonite, plant extracts.
4. The mycotoxin biodegrading agent of claim 3, further comprising a probiotic formulation including, but not limited to, one or more of Bacillus subtilis, Bacillus licheniformis, Bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp lactis, Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, Candida utilis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Streptococcus thermophilus, Lactobacillus reuteri, Bifidobacterium animalis, Aspergillus oryzae, Bacillus lentus, Bacillus pumilus, Lactobacillus cellobiosus, Lactobacillus fermentum, or Lactobacillus delbrueckii subsp, preferably, when used in silage or cattle feed, further comprises at least one of propionibacterium, lactobacillus buchneri and lactobacillus paracasei; or the mycotoxin biodegradation agent also contains bacillus coagulans and/or brevibacillus laterosporus when being used for feeds of poultry, pigs and aquaculture animals.
5. The mycotoxin biodegrading agent of claim 3, further comprising an additional enzyme, including but not limited to deoxynivalenol epoxide hydrolase, carboxypeptidase, aflatoxin oxidase, zearalenone lactonase, fumonisin carboxyesterase, fumonisin aminotransferase, laccase, aminopolyol amine oxidase, aspergillus niger aspartic protease, elastase, aminopeptidase, pepsin-like protease, trypsin-like protease, bacterial protease, amylase, arabinase, arabinofuranosidase, catalase, cellulase, hemicellulase, lignin degrading enzyme, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, galactosidase, etc, Glucanase, glucanotlyase, endoglucanase, glucoamylase, glucose oxidase, glucosidase, glucuronidase, hexose oxidase, hydrolase, invertase, isomerase, lipolytic enzyme, lyase, mannosidase, xylanase, oxidase, oxidoreductase, aldoketoreductase, pectate lyase, pectin acetylesterase, pectin depolymerase, pectin methylesterase, pectinolytic enzyme, peroxidase, manganese peroxidase, phenol oxidase, phytase, polygalacturonase, alcohol dehydrogenase, rhamnogalacturonase, ribonuclease, transferase, transporter, transglutaminase, epoxide hydrolase, hexose oxidase, acid phosphatase, and enzymes involved in starch metabolism, fiber degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism.
6. Use of a bacillus belgii according to claim 1 or a mycotoxin biodegradation agent according to any one of claims 2 to 5 for the detoxification of mycotoxins.
7. A method of degrading mycotoxins, comprising the steps of:
treating a mycotoxin-containing material with the Bacillus belgii of claim 1 or the mycotoxin biodegradation agent of any one of claims 2-5; the material includes, but is not limited to, one or more of food, feed and raw materials thereof, grain and by-products of processing thereof, grain oil, milk, aged grain, tea, fruits, fruit juices, or chinese herbal medicines.
8. Use of a method of degrading mycotoxins according to claim 7 for the detoxification of mycotoxins.
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