CN114214222A - High-temperature-resistant bacterial strain for efficiently degrading zearalenone and microbial inoculum thereof - Google Patents
High-temperature-resistant bacterial strain for efficiently degrading zearalenone and microbial inoculum thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention relates to a bacillus subtilis capable of resisting high temperature and efficiently degrading zearalenone and vomitoxin, which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC NO.22996, a microbial inoculum for efficiently degrading zearalenone and degrading vomitoxin and application thereof, wherein the main active component of the microbial inoculum is bacillus subtilis Bac.sp.Haut.1, the form of the microbial inoculum is solid powder, and a preparation method and application of the solid powder microbial inoculum are provided; the invention relates to a high-temperature-resistant bacterial strain for efficiently degrading zearalenone and a microbial inoculum thereof, and has the advantages of capability of simultaneously degrading zearalenone and vomitoxin and high degradation efficiency.
Description
Technical Field
The invention relates to a high-temperature-resistant bacterial strain for efficiently degrading zearalenone and application of a bacterial agent thereof in degrading zearalenone and vomitoxin in grains.
Background
Zearalenone (ZEN) is a nonsteroidal estrogen mycotoxin, also known as F-2 toxin, with the chemical name 6- (10-hydroxy-6-oxycarbenylene) -beta-clavulanic acid-mu-lactone. Is a lactone structure of dihydroxy benzoic acid of phenol, and is similar to the structure of animal endogenous estrogen beta-estradiol. Is a toxic secondary metabolite produced by fusarium such as fusarium tricuspidatum, fusarium moniliforme, fusarium graminearum, fusarium equiseti, fusarium roseum and the like. Zearalenone can contaminate food crops, such as corn, wheat and soybean, etc., causing problems with human food safety. Zearalenone can also contaminate the feed, causing animal poisoning. After animals eat a large amount of zearalenone polluted feed, toxic effects such as reproductive toxicity, cytotoxicity, genetic toxicity and immunotoxicity appear, and the generated toxicity influences the health of the animals and human beings through the inhibition effect on the reproductive function, the immune function and the like of an organism.
Deoxynivalenol (DON), also known as vomitoxin, is a trichothecene compound produced by Fusarium such as Fusarium graminearum, Fusarium equiseti, Fusarium roseum and the like, and is one of mycotoxins. The DON toxin can pollute food crops, cause food safety problems for human beings and cause animal poisoning. Vomitoxin has strong cytotoxicity and immunotoxicity, has great influence on the digestive system, the blood system and the nervous system of animals, and is listed as a tertiary carcinogen by the European Union.
Zearalenone toxin and emetic toxin are now ubiquitous in cereals and it is therefore of particular importance to find a suitable method for removing or reducing the toxicity of such toxins.
Contamination of food stuffs with ZEN and DON toxins can occur in agricultural fields, during transport and during processing, storage and consumption as food and feed products. In agricultural product samples in most areas of China, ZEN pollution is serious, the detectable rate is high, and because ZEN has extremely high thermal stability, the ZEN cannot be completely removed through conventional treatment. ZEN can therefore be detected in finished foods of cereals, such as bread or steamed bread. The Wutai pavilion and the like investigate the pollution condition of zearalenone in the grain processing process, and the detection result shows that the detection rate of ZEN toxin in the corn grinding processing reaches 34 percent; the detection rate of ZEN toxin in wheat flour exceeds 74 percent. DON has the advantages of extremely strong thermal stability, high detectable rate of DON in grains, and DON is absorbed in stomach and intestine, and harms various organs such as liver after entering a blood system, and can be completely absorbed 8h after entering a human body, and vomitoxin and metabolite thereof cannot be completely detected.
Hitherto, there are three major methods for detoxifying zearalenone toxin and vomitoxin at home and abroad, namely a physical method, a chemical method and a biological method. The physical method mainly comprises a radiation method, an adsorbent adsorption method, a heat treatment method and the like, and the chemical method mainly comprises the step of enabling ZEN and DON to react with alkali, an oxidant and an organic solvent to convert the ZEN and DON into other substances so as to reduce toxicity. The chemical methods at present mainly comprise a hydrogen peroxide treatment method, an ozone treatment method, a sodium carbonate soaking method and the like. Although physical and chemical methods can achieve a certain detoxification effect, they still have some disadvantages such as unsatisfactory detoxification effect, unclear detoxification products, possible loss of several important nutrients, chemical detoxification agent residues, high cost, and the like. The biological method can solve the problems in the physicochemical detoxification, and the biological method carries out the detoxification in a mild environment, thereby not only causing no loss of nutrient substances of food and feed, but also changing the palatability and bringing no toxic and harmful chemical substances. There are two current approaches to biological detoxification: the microbial adsorption detoxification has the advantages that firstly, the microbial adsorption detoxification has an action mechanism that bacteria cells adsorb toxins to form stable complexes, but the process is easily influenced by temperature and bacteria concentration and is reversible; and secondly, degrading and detoxifying the microorganisms or metabolites thereof, namely weakening the toxicity of ZEN and DON even degrading the ZEN and the DON into nontoxic substances by using intracellular enzymes, extracellular enzymes and secondary metabolites secreted by the microorganisms. And the microorganism has high propagation speed, various varieties and rich gene resources, and has huge potential for degrading mycotoxin. Thus, the biological method is the best method for degrading ZEN and DON in grains and feeds at present.
In conclusion, in order to guarantee food safety and body health of people, the problem of pollution of agricultural products and feeds ZEN and DON is solved. It is necessary to separate strains which can safely and efficiently degrade zearalenone and vomitoxin from natural resources, and enrich a resource pool which can simultaneously degrade zearalenone and vomitoxin degrading bacteria. Further research on its biological properties, toxin degradation properties and degradation mechanism. Provides technical support for the degradation of zearalenone and vomitoxin in grains.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a high-temperature-resistant bacterial strain for efficiently degrading zearalenone and a microbial inoculum thereof, and provides technical support for degrading zearalenone and vomitoxin in grains.
The invention is realized by the following technical scheme: a Bacillus subtilis (Bacillus subtilis) capable of resisting high temperature and efficiently degrading zearalenone and vomitoxin is named as Bac.sp.Haut.1, is separated from soil for planting corn in the spring Yang city of the Yongyang city of the Chongzhou province, 2021, 8 months and 2 days and is preserved in the China general microbiological culture Collection center (CGMCC) at the address: no. 3 of Xilu No. 1 of Beijing Korean district, the preservation number is CGMCC NO.22996, and like other biological cases, the bacterium with the activity of degrading zearalenone of the invention still has the possibility of mutation or variation of Bac.sp.Haut.1. Thus, mutants of this strain may be obtained by physical and chemical methods known in the art, for example, by treatment with chemical agents such as Nitrosoguanidine (NTG) and other chemical mutagens, or by physical methods such as ultraviolet, Co60 irradiation, and are also part of the present invention, provided that one of the characteristics of the ability to degrade zearalenone is retained.
The invention also provides a microbial inoculum, the active ingredient of which is Bacillus subtilis Bac.sp.Haut.1 (the preservation number is CGMCC NO.22996) or a mutant strain derived from the Bacillus subtilis. The microbial inoculum provided by the invention can be a liquid microbial inoculum or a solid microbial inoculum and is prepared by a preparation method disclosed in the prior art.
Further, bacillus subtilis Bac.sp.Haut.1 is extracellular enzyme having a degradation effect on zearalenone.
Further, after the bacillus subtilis Bac.sp.Haut.1 is treated at the high temperature of 85 ℃ for 20min, the viable count is 1 multiplied by 108-1×109CFU/mL。
A microbial inoculum for efficiently degrading zearalenone and vomitoxin mainly comprises a bacillus subtilis Bac.sp.Haut.1 in a solid powder form.
The invention also claims the application of the microbial inoculum in degrading zearalenone and vomitoxin in grains.
Further, the prepared microbial inoculum is added into grain, feed raw material and feed polluted by zearalenone and vomitoxin according to 0.5% -1%, and mixed, and the number of living cells is detected to reach 1 × 108-1×1010CFU/mL。
A method for preparing a solid powdery microbial inoculum comprises the following steps:
(1) activating and growing the bacillus subtilis Bac.sp.Haut.1 in an NA solid culture medium;
(2) selecting the bacillus subtilis Bac.sp.Haut.1 obtained in the step (1) to an NA liquid culture medium added with glucose, culturing for 48 hours at the pH of 7.0-7.5 and the fermentation condition of 27-37 ℃ and the rotating speed of 150-200 rpm to ensure that the number of viable bacteria in the fermentation liquid is 1 multiplied by 109-1×1011CFU/mL to obtain a liquid microbial inoculum;
(3) taking the mass of the liquid microbial inoculum as 1, adding 2-4% of starch, 1-2% of sodium metabisulfite and 1-2% of L cysteine hydrochloride protective agent, and performing spray drying to prepare the solid microbial inoculum.
Further, the conditions of spray drying were: the air inlet temperature is 175-.
Further, 800-9-1×1011CFU/mL) and 25-50 muL zearalenone with the concentration of 100 mug/mL for 6h, and the degradation rate of the zearalenone reaches more than 90 percent；
Further, 800-9-1×1011CFU/mL) and 25-50 muL of vomitoxin with the concentration of 100 mug/mL for 72 hours, and the degradation rate of the vomitoxin reaches more than 60 percent.
The invention has the beneficial effects that: according to the method for positioning and analyzing the active substances for degrading zearalenone by bacillus subtilis Bac.sp.Haut.1, the fermentation liquid is divided into cell-free supernatant, thallus suspension and intracellular extract, the degradation rate is respectively measured, and the main degradation active substances are concentrated in the cell-free supernatant. The cell-free supernatant was subjected to heat treatment, proteinase K treatment and proteinase K + SDS treatment, and the degradation activity of the cell-free supernatant was decreased, so that it was confirmed that the degradation active substance was mainly extracellular enzyme.
Drawings
FIG. 1 is a 48-hour degradation power curve of strain Bac.sp.Haut.1 degrading ZEN;
FIG. 2 shows the degradation rate of strain Bac.sp.Haut.1 cell-free supernatant, cell suspension and intracellular extract on ZEN and the degradation rate of three treatment groups after the cell-free supernatant is subjected to heat treatment, proteinase K treatment and proteinase K + SDS treatment;
FIG. 3 is a colony morphology of strain Bac.sp.Haut.1 cultured on NA medium for 24 h;
fig. 4 is an image collected by enlarging the strain bac.sp.haut.1 under an optical microscope 1000 times after gram staining;
FIG. 5 is a high performance liquid chromatogram of ZEN content before and after 12h of strain Bac.sp.Haut.1 degradation;
FIG. 6 is a high performance liquid chromatogram of DON content before and after 12h degradation of strain Bac.sp.Haut.1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
And (3) determining the degradation effect and the degradation active ingredients of the strain Bac.sp.Haut.1.
1. Activation of bacterial strains
The strain is inoculated in an inorganic salt culture medium (toxin concentration is 20ug/mL) coated with ZEN toxin for activation, and is placed in a 37 ℃ mould incubator for culturing for 48 h.
2. Fermentation of bacterial strains
And (3) selecting the activated strain to an NA culture medium containing glucose for fermentation, and performing shaking table fermentation at the temperature of 37 ℃ and at the speed of 150r/min for 48 h.
3. Degradation power curve of strain Bac.sp.Haut.1
Transferring 950 mu L of fermentation liquid into a 10mL centrifuge tube, adding 50 mu L of zearalenone toxin with the concentration of 100 mu g/mL, uniformly mixing by swirling for 30s, and setting the ZEN concentration in the degradation system to be 5 mu g/mL. Samples were taken at 6h, 12h, 24h, 36h and 48h after incubation, respectively, to determine the degradation rate of ZEN.
As a result, as shown in FIG. 1, the degradation rate of ZEN gradually increased with time, and at 6 hours, the degradation rate of ZEN reached 90% or more.
4. Degradation experiment of bacterial liquid, supernatant, bacterial suspension and intracellular extract of bacterial strain
Centrifuging the strain fermentation liquor for 10min at 4 ℃ and 8000r/min, transferring the centrifuged supernatant into a 10mL centrifuge tube, storing in a 4 ℃ refrigerator, washing the rest precipitated thallus with sterilized Phosphate Buffer Solution (PBS) for twice, and then re-suspending with PBS to obtain a bacterial suspension. Placing the bacterial suspension on ice, placing in an ultrasonic cell disruptor, performing ultrasonic treatment for 3s at intervals of 3s for 15min, then performing refrigerated centrifugation at 12000r/min for 20min, taking supernatant, and performing sterile suction filtration with a 0.22 μm suction filter to obtain filtrate, i.e. intracellular extract. Respectively taking 5mL of supernatant, carrying out high-temperature heating treatment at 121 ℃ for 20min to obtain a treatment group 1, adding proteinase K to obtain a concentration of 5mg/mL to obtain a treatment group 2, adding 5mg of proteinase K and 0.05g of SDS to obtain a treatment group 3, respectively taking 950 mu L of the supernatant, the bacterial suspension, the intracellular extract, the treatment groups 1, 2 and 3 and 50 mu L of ZEN toxin standard solution, placing the mixture in a sterilized 10mL centrifuge tube, and swirling for 30s to obtain a final ZEN concentration of 5 mu g/mL. The air bath shaking table is placed in the dark, the culture conditions are set to be 150r/min, 37 ℃ and 48h, and 3 groups of parallel experiments are carried out. After the reaction was completed, the degradation rate was measured.
The degradation effect of each fraction is shown in figure 2. The degradation rate of the strain on ZEN is mainly concentrated in cell-free supernatant, the thallus plays a certain adsorption role, and intracellular extracts basically have no degradation effect. After heating and treatment with proteinase K and proteinase K + SDS, the degradation rate of the supernatant was therefore initially concluded to be extracellular enzyme as the active ingredient for degrading ZEN.
Example 2
Identification of ZEN and DON high-efficiency degrading strain Bac.sp.Haut.1
1. Genomic DNA extraction
1) And (3) taking 1.5ml of a centrifuge tube, adding 200 mu L of pretreatment liquid and three glass beads, adding an appropriate amount of a bacteria sample, and putting the bacteria sample into a grinding instrument for grinding fully.
2) Adding 20 mu LProteinase K solution, mixing uniformly, and standing at 37 ℃ for 30-60 min.
3) Add 200. mu.L of lysis buffer, mix well by inversion, and stand at 70 ℃ for 10 min.
4) Add 200. mu.L of absolute ethanol, mix well by inversion, and centrifuge briefly to remove droplets on the inner wall of the tube cap.
5) Passing through adsorption column, washing with washing solution for 1 time, and washing with rinsing solution for 2 times.
6) And (5) placing the adsorption column at room temperature for 5-10 minutes to thoroughly dry the residual rinsing liquid in the adsorption material.
7) Transferring the adsorption into a new centrifuge tube, suspending and dropwise adding 50-100 μ L ddH 2O to the middle position of the adsorption film, standing at room temperature for 5-10min, centrifuging at 12,000 rpm for 2min, and collecting the solution into the centrifuge tube.
2.16S amplification
Table 2: primer information
Table 3: PCR amplification reaction system and conditions
PCR product detection and purification
mu.L of the PCR product was subjected to 1.0% agarose gel detection, and the band property was observed.
And (3) purifying the PCR product according to the operation of a magnetic bead purification standard operation flow, adsorbing DNA in a high-salt low-pH solution by utilizing the principle that magnetic beads can adsorb or release substances with charges, and releasing DNA in a low-salt high-pH solution, so that the aim of separating and purifying the DNA product is fulfilled.
4. Sequencing
And (4) performing on-machine detection on the purified PCR product.
5. Comparison of results
The sequencing results were aligned with NCBI-BLAST.
The comparison results are shown in Table four, and the identification result of the 16s DNA of the strain is Bacillus (Bacillus) Table 4 BLAST comparison results
6. Physiological and biochemical identification
Strain bac.sp.haut.1 was streaked onto the fermentation medium and observed for colony morphology as in fig. 3, colonies were picked for gram staining and observed using an optical microscope as in fig. 4. Sterile operation, selecting pure culture of separated strain, inoculating in biochemical reaction tube, culturing at 37 deg.C for 48 hr, and measuring 11 physiological and biochemical indexes such as sugar, salt, MR, VP, indole experiment, etc. The results were observed and recorded, and the results are shown in table 4. The physiological and biochemical characteristics show that the strain is bacillus subtilis as shown by the final identification result.
Table 5 physiological and biochemical identification of strain bac.sp.haut.1
Note: "+" is a positive reaction and "-" is a negative reaction.
Example 3
High temperature resistance test of strains
The fermentation broth obtained as described in example 1 was subjected to a water bath at 85 ℃ for 20min, and the heated fermentation broth was diluted with sterile physiological saline to a final dilution ratio of 10-7、10-8And 10-9. And (3) coating the diluent into an NA solid culture medium, carrying out inverted culture at 37 ℃ for 12h, observing the growth condition of the flat plate with each concentration, and calculating the viable count.
The number of viable bacteria can still reach 1 × 10 after the fermentation liquor is subjected to water bath at 85 ℃ for 20min8-1×109CFU/mL. Therefore, the strain can endure high temperature, can ensure higher viable count under a short-time high-temperature processing environment, and does not influence the subsequent degradation effect of the strain.
Example 4
Preparation test of bacterial preparation
1. Activation of bacterial strains
The strain is inoculated in an inorganic salt culture medium (toxin concentration is 20ug/mL) coated with ZEN toxin for activation, and is placed in a 37 ℃ mould incubator for culturing for 48 h.
2. Bacterial strain seed liquid
Selecting the activated strain to NA culture medium containing glucose for fermentation, and performing shake fermentation at 37 deg.C and 150r/min for 24h to make OD value reach 1 × 108CFU/mL。
3. Fermentation culture
The seed solution was inoculated into a 30L jar fermentor at an inoculum size of 4%, and the glucose NA-containing medium in the jar fermentor was autoclaved at 121 ℃ for 20 min. The fermentation conditions were: pH 7.0-7.5, temperature 37, stirring speed 180rpm, dissolved oxygen amount 60%, and culture time 36-50 h until viable count in fermentation tank reaches 1 × 109-1×1011CFU/mL。
4. Spray drying
Mixing wheat bran and corncob powder according to any proportion to form a base material, and mixing the produced fermentation liquor with the base material according to a mass ratio of 1: 5, mixing uniformly, adding 2-4% of starch, 1-2% of sodium metabisulfite and 1-2% of L-cysteine hydrochloride protective agent according to the weight ratio, and carrying out spray drying to prepare the solid microbial inoculum. The conditions of spray drying were: the air inlet temperature is 175-.
5. Application of microbial inoculum
Mixing 950 mu L of the prepared liquid microbial inoculum with 50 mu L of zearalenone with the concentration of 100 mu g/mL, co-culturing for 12h, and determining the content of zearalenone toxin.
And (3) extraction and detection of zearalenone: extracting zearalenone toxin of the mixed solution by adopting dichloromethane with three times volume, slowly blowing the extract at 35 ℃ by using nitrogen, re-dissolving 1mL of methanol after blowing, filtering by using a 0.22 mu m filter membrane, pumping into a sample injection bottle, and detecting the toxin by using a high performance liquid chromatography;
the degradation effect is shown in fig. 5, the content of zearalenone before and after degradation can be clearly compared, the toxin peak time is 5.09min, and the degradation rate reaches 95.3%.
Mixing 950 μ L of the prepared liquid microbial inoculum with 50 μ L of vomitoxin with concentration of 100 μ g/mL, co-culturing for 72h, and determining vomitoxin content.
Extraction and detection of vomitoxin: extracting the vomitoxin in the reacted bacterial liquid by using a vomitoxin immunoaffinity column, and detecting the toxin by using a high performance liquid chromatography after extraction.
The degradation effect is shown in fig. 6, the content of vomitoxin before and after degradation can be clearly compared, the toxin peak time is 6.6min, and the degradation rate reaches 65.6%.
The prepared solid microbial inoculum is added into grain, feed raw material and feed polluted by zearalenone according to 0.5% -1%, and is uniformly mixed, and the number of living cells is detected to reach 1 × 108-1×1010CFU/mL。
In the prior art, "a strain of bacillus amyloliquefaciens and application thereof in degrading zearalenone" (Chinese patent publication No. CN103981133A), the strain of bacillus amyloliquefaciens is cultured for 24 hours under a proper condition, and the zearalenone with a final concentration of 5 mug/ml is added into an MM culture medium for co-culture for 72 hours, wherein the degradation rate is 95.99%. In the bacillus subtilis for efficiently degrading zearalenone and the application thereof (Chinese patent publication No. CN105385616A), the bacillus subtilis is cultured for 6 hours together with 20 mu g/mL zearalenone after being cultured under a proper condition, and the degradation rate is 100 percent;
compared with the prior art, the strain is cultured for 48 hours under proper conditions and then co-cultured with zearalenone for 24 hours, the degradation rate is more than 97%, and the strain is co-cultured with vomitoxin for 72 hours while the zearalenone is efficiently degraded, so that the degradation rate of the vomitoxin can also be more than 60%.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (10)
1. A bacillus subtilis capable of resisting high temperature and efficiently degrading zearalenone and vomitoxin is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC NO. 22996.
2. The bacillus subtilis Bac.sp.Haut.1 capable of resisting high temperature and efficiently degrading zearalenone and vomitoxin according to claim 1, wherein the bacillus subtilis Bac.sp.Haut.1 has an extracellular enzyme with a zearalenone degradation effect.
3. The bacillus subtilis Bac.sp.Haut.1 capable of resisting high temperature and efficiently degrading zearalenone and vomitoxin according to claim 1, wherein the viable count of the bacillus subtilis Bac.sp.Haut.1 is 1 x 10 after the bacillus subtilis Bac.sp.Haut.1 is subjected to high-temperature treatment at 85 ℃ for 20min8-1×109CFU/mL。
4. The microbial inoculum for efficiently degrading zearalenone and vomitoxin is characterized in that the active ingredient of the microbial inoculum is bacillus subtilis Bac.sp.Haut.1, and the form of the microbial inoculum is solid powder.
5. The use of the microbial inoculum of claim 4 in degrading zearalenone and vomitoxin in foodstuffs.
6. The use as claimed in claim 5, wherein the prepared microbial inoculum is added to grain, feed raw material or feed contaminated by zearalenone and vomitoxin in an amount of 0.5% -1%, mixed, and the number of viable cells is detected to be 1 x 108-1×1010CFU/mL。
7. A method for preparing the solid powdery microbial inoculum according to claim 4, which comprises the following steps:
(1) activating bacillus subtilis Bac.sp.Haut.1 in an NA solid culture medium until a single colony grows out;
(2) selecting a single colony of the bacillus subtilis Bac.sp.Haut.1 obtained in the step (1) to be added into an NA liquid culture medium added with glucose, wherein the pH is 7.0-7.5, the fermentation condition is that the temperature is 27-37 ℃, the rotating speed is 150rpm-200rpm, and the culture is carried out for 48 hours, so that the viable count in the fermentation liquor is 1 multiplied by 109-1×1011CFU/mL to obtain a liquid microbial inoculum;
(3) adding 2-4% of starch, 1-2% of sodium metabisulfite and 1-2% of L cysteine hydrochloride protective agent into the prepared liquid microbial inoculum, and carrying out spray drying to prepare the solid microbial inoculum.
8. The method for preparing a solid powdery microbial agent according to claim 7, wherein the spray-drying conditions are as follows: the air inlet temperature is 175-.
9. The application of the liquid bacterial agent as claimed in claim 7, wherein the viable count of 800-9-1×1011The fermentation liquor of CFU/mL and zearalenone with the concentration of 100 mu g/mL are co-cultured for 6 hours, the zearalenone is degraded, and the degradation rate of the zearalenone reaches more than 90%.
10. The application of the liquid bacterial agent as claimed in claim 7, wherein the viable count of 800-9-1×1011The CFU/mL fermentation liquid and 25-50 mu L vomitoxin with the concentration of 100 mu g/mL are co-cultured for 72 hours, and the degradation rate of the vomitoxin reaches more than 60 percent.
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| CN111808765A (en) * | 2019-12-27 | 2020-10-23 | 贾如 | Bacillus subtilis capable of efficiently degrading vomitoxin and application thereof |
| CN112410269A (en) * | 2020-12-09 | 2021-02-26 | 中国科学院天津工业生物技术研究所 | Bacillus subtilis and application thereof in degradation of zearalenone |
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| CN105385616A (en) * | 2015-09-02 | 2016-03-09 | 国家粮食局科学研究院 | Bacillus subtilis for effectively degrading zearalenone and application thereof |
| CN111808765A (en) * | 2019-12-27 | 2020-10-23 | 贾如 | Bacillus subtilis capable of efficiently degrading vomitoxin and application thereof |
| CN112410269A (en) * | 2020-12-09 | 2021-02-26 | 中国科学院天津工业生物技术研究所 | Bacillus subtilis and application thereof in degradation of zearalenone |
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