CN115948305A - Bacillus subtilis capable of efficiently degrading various mycotoxins and application thereof - Google Patents
Bacillus subtilis capable of efficiently degrading various mycotoxins and application thereof Download PDFInfo
- Publication number
- CN115948305A CN115948305A CN202310082062.0A CN202310082062A CN115948305A CN 115948305 A CN115948305 A CN 115948305A CN 202310082062 A CN202310082062 A CN 202310082062A CN 115948305 A CN115948305 A CN 115948305A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- strain
- culture
- solution
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention belongs to the technical field of microbial preparations and application of natural products, and particularly relates to bacillus subtilis for efficiently degrading various mycotoxins and application thereof. The invention provides an active antibacterial lipopeptide high-producing strain, which is bacillus subtilis. The invention also provides application of the bacillus subtilis in preparation of the antibacterial lipopeptide, and the bacillus subtilis is used for preparing and producing the antibacterial lipopeptide by a biological fermentation method. Compared with the prior art, the bacillus subtilis and the secreted antibacterial lipopeptide active substances obtained by screening can simultaneously and efficiently degrade at least three mycotoxins, and the problem that a single feed strain only acts on one mycotoxin at the present stage and is difficult to practically apply due to limited application range is solved. The strain can be used for developing novel biological detoxifiers, feed additives and the like, is very suitable for being used as feed additives, and has good application value and market popularization prospect in feed production and breeding industries.
Description
Technical Field
The invention belongs to the technical field of application of microbial preparations and natural products, and particularly relates to bacillus subtilis for efficiently degrading various mycotoxins and application thereof, in particular to high-yield bacillus subtilis with active antibacterial lipopeptide, and application of the high-yield strain and the antibacterial lipopeptide in degradation of mycotoxins.
Background
The quality safety of the feed is the first gateway for ensuring the safety of cultured products and food safety, and the prevention of feed mildew becomes a common concern of the feed industry. Currently, at least 25% of the grains in the world are contaminated with mycotoxins, as estimated by the Food and Agriculture Organization (FAO) of the united nations. The pollution condition of mycotoxin in China is not optimistic, and the pollution rate of mycotoxin reaches 90 percent. Among the various mycotoxins, the most harmful are aflatoxins, zearalenone (F2 toxin), and vomitoxin and trichothecenone (T2 toxin).
1088 feed samples from different regions of China are detected by a report on the detection of feed and raw material mycotoxins from the Ministry of Changchang research of Shandong Longchang, and the coexistence of various toxins in all samples is obvious, wherein 5 percent of samples with 1 mycotoxin detected, 20 percent of samples with 2 mycotoxins detected and 75 percent of samples with 3 or more mycotoxins detected. Aflatoxins B in different types of samples 1 The positive detection rates of zearalenone and vomitoxin are all more than 87%.
Mycotoxins are toxic secondary metabolites produced by fungi (molds). Aflatoxins are produced by aspergillus flavus, and can cause liver necrosis, reduce production efficiency, reduce milk yield, cause congenital defects, tumors and inhibit the immune system. Zearalenone toxin and vomitoxin are both produced by fusarium, have various strong toxicities and are extremely harmful to animals and human bodies. Zearalenone and its metabolites can cause hyperestrogenism and reproductive disorders in a wide variety of female animals, for example, feeding contaminated zearalenone feed can cause female pigs to produce estrogenic toxicosis, resulting in a series of reproductive disorders such as pseudopregnancy, infertility, ovarian malformation, and abortion. Vomitoxin can affect the digestive system, blood system, central system and calcium and phosphorus metabolism of animals. Low doses of emetic toxin in the feed can cause symptoms such as loss of appetite, weight loss, metabolic disturbances, etc. in animals, high doses can cause vomiting of the animal, pigs being the most sensitive animal to emetic toxin.
A large number of researches show that mycotoxins widely exist in grains, animal feeds and agricultural and sideline products, and particularly, the pollution condition of zearalenone, aflatoxin and vomitoxin in food processing or feed production taking corns, wheat, rice, peanuts and the like as raw materials is the most serious. Although some processes (e.g., high temperature tempering) can kill some microorganisms such as mold, the toxins produced by mold metabolism are difficult to eliminate. After the mycotoxin pollutes the feed, the mycotoxin can not only directly harm the health of animals and human health, but also limit the application of grains in food, medicine, feed and deep processing industries. In the prior art, methods such as montmorillonite adsorption and enzymolysis are mostly adopted to eliminate mycotoxin pollution, but the effects are not ideal and the cost is very high.
In recent years, the adoption of the microbial probiotics and the generated active substances as feed additives plays a key role in preventing mildew of feeds. However, the currently used probiotic additives for feeding mainly only have a degradation effect on a single mycotoxin, for example, a strain of saccharomyces zearalenone degrading yeast and an application thereof (CN 105794963A), a bacillus subtilis (CN 101705203B) for degrading aflatoxin, an enterococcus faecium gxsccu 1 for efficiently degrading vomitoxin, a microbial agent and a preparation method and an application thereof (CN 114437983A) in chinese patent.
Feed is often affected by various mycotoxins due to the influence of raw material sources and storage environments, and a microorganism capable of simultaneously and irreversibly degrading various mycotoxins has not been found so far. According to the invention, a bacillus subtilis strain is obtained through screening, and secondary metabolites of the bacillus subtilis strain are extracted and analyzed, so that the bacillus subtilis and the active antibacterial lipopeptide secreted by the bacillus subtilis strain can efficiently degrade at least three mycotoxins at the same time, and the bacillus subtilis strain has high detoxifying activity, strong specificity and mild action effect, and has wide application prospects in the field of feed additives.
Disclosure of Invention
The mycotoxin pollutes cereals and feeds in a wide range and is harmful greatly, and the problem of low degradation efficiency exists when the microorganisms capable of degrading the mycotoxin are singly used as microbial agents or feed additives are prepared at the present stage, so that the mycotoxin pollution is not suitable for actual production. Therefore, aiming at the defects in the prior art, the invention provides a beneficial microorganism (bacillus subtilis), the microorganism and the secreted active metabolite (antibacterial lipopeptide) thereof can efficiently degrade a plurality of mycotoxins in feed or processing raw materials, especially can reduce the toxicity of aflatoxin, zearalenone and vomitoxin to human beings or animals, improve the nutritive value of feed or food processing raw materials, and lay a foundation for developing an efficient, safe and environment-friendly microecological preparation capable of degrading the mycotoxins.
Furthermore, the invention also aims to provide application of the bacillus subtilis in preparation of antibacterial lipopeptide.
Further, the invention also aims to provide application of the bacillus subtilis or the antibacterial lipopeptide in degrading various mycotoxins.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
an active antibacterial lipopeptide high-yield strain is Bacillus subtilis, and is obtained by screening and separating deep sea mud in a deep sea culture base of the sea cucumber in the great continuous sea; for convenience of recording and management, the inventors named it: bacillus subtilis BYA-KC-4; the morphological characteristics of the strain are as follows: the bacterial colony is circular, smooth in surface, raised, opaque, milky white, neat in edge, certain in viscosity and l-2 cm in diameter; gram positive staining; 0.6-0.9 (width) multiplied by 1.0-1.5 μm (width), oval or oval, and no obvious expansion of the thalli after the spores are formed.
The strain is deposited by the inventor at 9/19/2022 at the deposition address: the microbial research institute of China academy of sciences, no. 3 of Xilu No.1 of Beijing, chaoyang, china Committee for culture Collection of microorganisms, general microbiological center (CGMCC), the preservation number of the strain is as follows: CGMCC No.25758.
Furthermore, the invention also provides application of the bacillus subtilis in preparing the antibacterial lipopeptide, and the antibacterial lipopeptide is prepared by a biological fermentation method.
Further, the invention also provides a method for producing the antibacterial lipopeptide by using the bacillus subtilis through fermentation, which comprises the following steps:
(1) Activating strains: inoculating bacillus subtilis into an LB solid culture medium, and culturing at 28-32 ℃ for 12-24 h to obtain an activated strain;
(2) Seed culture: inoculating the activated strain to an LB liquid culture medium, and culturing at 28-32 ℃ for 16-18 h to obtain a first-stage seed solution;
(3) And (3) expanding culture: inoculating the first-stage seed liquid to an LB liquid culture medium, and culturing at 28-32 ℃ for 16-18 h to obtain a spread culture seed liquid;
(4) Fermentation culture: inoculating the expanded culture seed solution to a fermentation culture medium, and culturing at 28-32 ℃ for 12-48 h to obtain fermentation liquor;
(5) Centrifuging the fermentation liquor obtained in the step (4) to obtain fermentation supernatant, adjusting the pH of the fermentation supernatant to 2.5 by using acid liquor to generate precipitate, standing for 2-24 hours to completely precipitate, discarding part of supernatant to obtain suspension, and centrifuging to obtain precipitate;
(6) And (3) extracting the precipitate obtained in the step (5) by using absolute ethyl alcohol to obtain an extracting solution, filtering to remove impurities, and distilling the ethanol solvent under reduced pressure at the temperature of 50-60 ℃ to obtain a solid product, namely the antibacterial lipopeptide.
Specifically, the LB liquid culture medium comprises the following components: in each liter of the medium, 10.0g of tryptone, 5.0g of yeast extract, and 10.0g of NaCl; when in use, the pH value is adjusted to 7.0-7.2, and the mixture is sterilized for 20min at 121 ℃.
The LB solid culture medium is prepared by adding agar powder into a liquid culture medium; the specific composition is that in each liter of culture medium, 10.0g of tryptone, 5.0g of yeast extract, 10.0g of NaCl and 18.0g of agar powder; when in use, the pH value is adjusted to 7.0-7.2, and the mixture is sterilized for 20min at 121 ℃.
Specifically, the fermentation medium comprises the following components: in each liter of the medium, 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of NaCl, K 2 HPO4 0.5g，MgSO 4 0.1g，MnSO 4 0.01g，FeSO 4 0.001g; when in use, the pH is adjusted to 7.0, and the mixture is sterilized at 121 ℃ for 20min.
Specifically, the amount of the inoculum (volume fraction) for the scale-up culture in step (3) is 1-2%.
Specifically, the concentration of the seed liquid obtained in the step (3) is 1x10 9 cfu/mL～2x10 9 cfu/mL。
Specifically, the inoculation amount (volume fraction) of the fermentation culture in the step (4) is 2-5%.
Specifically, the acid solution in the step (5) is a hydrochloric acid solution with the concentration of 5-8 mol/L.
Specifically, the solid-to-liquid ratio of the precipitate to absolute ethyl alcohol in the step (6) is 1g.
Further, based on a general inventive concept, the present invention also provides the use of said bacillus subtilis for the degradation of mycotoxins.
Further, based on a general inventive concept, the present invention also provides the use of the antimicrobial lipopeptides for the degradation of mycotoxins.
Specifically, the antimicrobial lipopeptides are capable of simultaneously degrading a plurality of mycotoxins, including but not limited to zearalenone, vomitoxin, aflatoxin B 1 。
Further, based on a general inventive concept, the present invention also provides a method for degrading mycotoxins using the antibacterial lipopeptide, comprising the steps of:
diluting the prepared antibacterial lipopeptide, centrifuging to obtain supernatant, mixing the supernatant with a mycotoxin solution for reaction, adjusting the reaction system to be acidic, and reacting for 2-72 h at 35-38 ℃.
Specifically, the pH of the reaction system was adjusted to 6.2 during the reaction.
Specifically, the mycotoxin is zearalenone toxin, vomitoxin and/or aflatoxin B 1 。
Specifically, the mycotoxin solution is zearalenone aqueous solution, vomitoxin aqueous solution and aflatoxin B 1 One, two or three of the aqueous solutions.
Specifically, the concentration of the supernatant is 50-100 mug/mL.
Specifically, the concentration of the mycotoxin solution is 200-10000 ppb.
Specifically, the volume ratio of the supernatant to the mycotoxin solution is (3-12): 1.
preferably, the volume ratio of the supernatant to the mycotoxin solution is 10:3 or 4.
Further preferably, the volume of the supernatant is 1000-1200 μ L, and the volume of the mycotoxin solution is 100-300 μ L.
Furthermore, the invention also provides application of the bacillus subtilis or the antibacterial lipopeptide in preparing a mycotoxin degradation medicament.
Furthermore, the invention also provides application of the bacillus subtilis or the antibacterial lipopeptide in preparing feed additives for degrading mycotoxin.
Specifically, the bacillus subtilis is prepared into bacterial powder which is used as a feed additive to be added into feed, and the specific addition amount is 3-5 g added in each kilogram of feed.
Further, the invention also provides a fungicide for degrading mycotoxin, wherein the active ingredients of the fungicide comprise bacillus subtilis BYA-KC-4 or mutant strains derived from the bacillus subtilis BYA-KC-4, and the preservation number of the bacillus subtilis BYA-KC-4 is as follows: CGMCC No.25758.
Specifically, the microbial inoculum is a liquid microbial inoculum or a solid microbial inoculum.
Specifically, the bacterial concentration of the bacillus subtilis BYA-KC-4 in the microbial inoculum is 1 multiplied by 10 10 ～3×10 10 cfu/mL。
Further, the invention also provides a preparation method of the mycotoxin degrading microbial inoculum, which comprises the following steps:
1) Activating strains: inoculating bacillus subtilis into an LB solid culture medium, and culturing at 28-32 ℃ for 12-24 h to obtain an activated strain;
2) Seed culture: inoculating the activated strain to an LB liquid culture medium, and culturing at 28-32 ℃ for 16-18 h to obtain a first-stage seed solution;
3) And (3) amplification culture: inoculating the first-stage seed liquid to an LB liquid culture medium, and culturing at 28-32 ℃ for 16-18 h to obtain a spread culture seed liquid;
4) Fermentation culture: inoculating the expanded culture seed liquid to a fermentation culture medium, and culturing at 28-32 ℃ for 12-48 h to obtain a fermentation liquid, namely the liquid microbial inoculum.
Specifically, the inoculation amount (volume fraction) of the amplification culture in the step 3) is 1-2%.
Specifically, the concentration of the seed liquid obtained in the step 3) is 1x10 9 cfu/mL～2x10 9 cfu/mL。
Specifically, the inoculation amount (volume fraction) of the fermentation culture in the step 4) is 2-5%.
Preferably, the microbial inoculum is a composite microbial inoculum, and the microbial inoculum comprises bacillus subtilis, clostridium butyricum, lactobacillus plantarum and saccharomyces cerevisiae; the dosage ratio of each bacterium is 3.
Compared with the prior art, the invention has the beneficial effects that:
1. the strain with remarkable inhibition effect on aspergillus flavus is obtained by repeated screening, and the strain can secrete high-yield active antibacterial lipopeptide; through liquid chromatography and mass spectrometry, the main component of the antibacterial lipopeptide secreted by the strain is Bacillomycin D (Bacillus D). Research shows that bacillomycin D is the only secondary metabolite which is obtained from microorganisms and identified to have resistance to aspergillus flavus and a plurality of moulds and mycotoxin, so that the strains screened by the invention can be used for degrading a plurality of mycotoxins.
2. Tests prove that the screened active antibacterial lipopeptide secreted by the bacillus subtilis BYA-KC-4 has the degradation rate of over 90 percent on zearalenone toxin, the degradation rate of over 80 percent on vomitoxin and the degradation rate on aflatoxin B 1 The degradation rate of the compound is more than 80 percent, and the compound has the capability of efficiently degrading various mycotoxins.
3. The bacillus subtilis BYA-KC-4 can also secrete lipase, amylase, protease and other enzymes, the enzyme activity is high, the digestive enzymes are very beneficial to the health of cultured animals, and the bacillus subtilis BYA-KC-4 is suitable to be used as an animal feed additive or microbial inoculum to promote the growth and development of animals and improve the feed utilization rate.
4. The bacillus subtilis BYA-KC-4 also has good stress resistance, can tolerate strong acid and strong alkali, has good thermal stability and pH stability, and is suitable for being added into a feed processing process to directly produce probiotic feed.
5. The bacillus subtilis BYA-KC-4 has multiple excellent characteristics, is very suitable to be used as a feed additive, can efficiently degrade various mycotoxins and secrete various digestive enzymes, has strong stress resistance, and is non-toxic and harmless to animals. Therefore, the strain is used for developing novel biological detoxifiers, feed additives and the like, reduces the pollution of mycotoxin to feed or processing raw materials, improves the feed utilization rate and the nutritional value, and has good application value and market popularization prospect in feed production and breeding industries.
Drawings
FIG. 1 shows the bacteriostatic results of the rescreening of example 1;
FIG. 2 is a bacteriostatic diagram of the effect of the active antibacterial lipopeptide produced by the strain BYA-KC-4 obtained by rescreening in example 1 on Aspergillus flavus; wherein, the label 1 is ethanol contrast, and the label 2 is ethanol solution of active antibacterial lipopeptide;
FIG. 3 is a liquid chromatogram of example 1, in which secondary metabolites produced by strain BYA-KC-4 were analyzed using a LC MS;
FIG. 4 is a graph showing the measurement of genetic stability at passage of strain BYA-KC-4 in example 1 of the present invention;
FIG. 5 is a colony morphology of strain BYA-KC-4;
FIG. 6 is a graph showing the time course of degradation of zearalenone toxin by the active antibacterial lipopeptide produced by Bacillus subtilis BYA-KC-4 in example 4 of the present invention;
FIG. 7 is a graph showing the time course of the activated antibacterial lipopeptide produced by Bacillus subtilis BYA-KC-4 in example 5 of the present invention degrading emetic toxin;
FIG. 8 shows that the active antibacterial lipopeptide produced by Bacillus subtilis BYA-KC-4 in example 6 of the present invention degrades aflatoxin B 1 Curve over time.
Detailed Description
For a better understanding of the present invention, the present invention is further described below with reference to the following examples, but the present invention is not limited to the following examples, and modifications and equivalents thereof without departing from the spirit of the present invention are included in the scope of the present invention.
The media and reagents involved in the examples are as follows:
LB solid medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of NaCl, 18.0g of agar powder and 1.0L of deionized water; the pH value is 7.0-7.2, and the sterilization is carried out for 20min at the temperature of 121 ℃.
LB liquid medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of NaCl, and adding deionized water to 1.0L; the pH value is 7.0-7.2, and the sterilization is carried out for 20min at the temperature of 121 ℃.
PDA culture medium: 200.0g of potato, 20.0g of glucose and 20.0g of agar, and adding deionized water to 1.0L; sterilizing at 115 deg.C for 30min under natural pH.
Fermentation medium: 10.0g of glucose, 10.0g of bovine bone peptone, 5.0g of NaCl, K 2 HPO4 0.5g，MgSO 4 0.1g，MnSO 4 0.01g，FeSO 4 0.001g, deionized water is added to 1.0L; sterilizing at 121 deg.C for 20min and pH 7.0.
Preparation of sterile physiological saline: 8.5g of NaCl is dissolved in a small amount of distilled water, and then the distilled water is added until the volume is 1000mL; sterilizing at 121 deg.C for 20min.
Preparation of PBS (0.01 mol/L) buffer: naCl 8g, KCl 0.2g, na 2 HPO 4 1.44g，KH 2 PO 4 0.24g of the extract is dissolved in 800mL of distilled water, the pH value of the solution is adjusted to 7.5 by NaOH, and finally the distilled water is added to the solution to be constant volume of 1L; sterilizing at 121 deg.C for 20min.
Example 1 screening method for Strain
Example 1 provides a method for screening bacillus subtilis with mycotoxin resistance, which comprises the following specific steps:
1. separation of the strains:
taking 1g of sea mud sample from deep sea mud in a deep sea culture base of sea cucumber in the great sea of great sea, adding 10mL of sterile physiological saline, fully shaking and uniformly mixing to obtain a sample solution, and performing gradient dilution (10) on the sample solution -4 ，10 -5 ，10 -6 ，10 -7 ) Then 100. Mu.L of each dilution was spread on an LB solid medium plate, cultured at 37 ℃ for 24 hours, and each dilution was repeated three times, and then a single colony was selected and purified.
2. Screening of anti-aspergillus flavus bacterial strain
2.1 culture of Aspergillus flavus and preparation of spore liquid: inoculating aspergillus flavus stored in a laboratory to a PDA inclined plane, and culturing for 6 days at 28 ℃; adding into the mixture after the spores are fully formedGently scraping off Aspergillus flavus spores on the inclined plane with 4mL of sterile normal saline to obtain spore suspension, transferring into a new sterile tube, sufficiently shaking, counting with a blood counting chamber, and adjusting the concentration to 10 7 The concentration is/mL, namely the mould spore liquid, and the mould spore liquid is stored at 4 ℃ for later use; the Aspergillus flavus is a commercially available product, and the specific strain number is Aspergillus flavus ATCC28539; the bacterial colony of the aspergillus flavus is yellow green and loose, and the hyphae are white and transparent, have partitions and branches and are loose and radial;
2.2 preliminary screening of the anti-aspergillus flavus strain: uniformly coating 100 mu L of the mould spore liquid obtained in the step 2.1 on a PDA (personal digital assistant) plate; then, dibbling the bacterial colonies obtained by separation and purification in the step (1) on plates, wherein the dibbling quantity of each plate is moderate, and two bacterial colonies are parallel; culturing at 28 deg.C for 48h, observing whether there is a bacteriostatic zone around the test bacterial colony, and screening to obtain primary screened strains, wherein the primary screened strains have good bacteriostatic effects, and are respectively numbered as B-301 and B-332;
2.3 re-screening of the anti-aspergillus flavus strain: inoculating the bacterial strain with bacteriostatic activity obtained by primary screening in the first loop step 2.2 by using an inoculating loop, inoculating the bacterial strain into an LB liquid culture medium, carrying out shaking culture at 37 ℃ and 180r/min for 24h, centrifuging the culture solution for 10min at 8000r/min, and collecting the supernatant for later use;
uniformly coating 100 mu L of the mould spore liquid prepared in the step 2.1 on a PDA (personal digital assistant) flat plate, placing three Oxford cups on each flat plate, and respectively preparing a test group added with 100 mu L of the supernatant liquid obtained in the step 2.3 and a control group added with 100 mu L of physiological saline; and finally, culturing all the plates in an incubator at 28 ℃ for 48-72 hours, observing whether the bacteria inhibition zone exists around the tested strains or not, and measuring the diameter of the bacteria inhibition zone to obtain a re-screening result, wherein the specific re-screening result is shown in table 1 and figure 1, and the bacteria inhibition zone of the strain with the number of B-332 is larger.
TABLE 1
3. Screening of antibacterial lipopeptide high-producing strains
Taking the strain (number is B-332) with the largest inhibition zone screened out again in the step 2.3 of one loop by using an inoculating loop, inoculating the strain into an LB liquid culture medium, carrying out shaking culture at 37 ℃ and 180r/min for 18h, then inoculating the strain into a fermentation culture medium according to 2 percent of inoculation amount (volume fraction), and carrying out fermentation culture at 30 ℃ and 180r/min for 48h; centrifuging fermented liquid, collecting supernatant, adding 10 mu L hydrochloric acid solution with the concentration of 6mol/L into 1mL of supernatant, adjusting the pH to 2-2.5, stirring and mixing uniformly, finding that a precipitate is generated, and standing at 4 ℃ for 12 hours; the precipitate was collected by centrifugation and dissolved in methanol, and then filtered with a 0.2 μm filter, and the filtered filtrate was analyzed for components using a liquid chromatography mass spectrometer (high performance liquid chromatography/2695 control panel 2998PDA, waters, usa), wherein the liquid chromatography conditions were: sample introduction volume: 15 mu L of the solution; column temperature: 30 ℃; DAD detector detection range: 190-640nm; detection wavelength: 200nm; mobile phase: ultrapure water and acetonitrile, wherein the A term is ultrapure water, and the B term is acetonitrile; the gradient elution conditions were: 0-1min, 5-B-5%; 1-20min, 5-95% by weight B;20-25min, keeping 95%; the mass spectrum conditions are as follows: an ion source: electrospray ionization (ESI); temperature of atomized gas: 350 ℃; spraying pressure: 35psig; positive and negative ion capillary voltage: 3500V; flow rate of drying gas: 8L/min; ion scanning range (m/z): 100-2000;
the components in the filtrate are subjected to structural identification, and through document (1, li Baoqing, luxiyun, guo Geng, and the like. The separation and identification of lipopeptides produced by Bacillus subtilis BAB-1 and volatile substances [ J ]. Chinese agriculture science 2010,43 (17): 3547-3554.;2, jasim B, sreelakshmi K S, mathew J, et al. Surfactin, iturin, and fengycin biosynthesis by hydrophylic Bacillus sp.frenopsis monnieri [ J ]. Microbiological Ecology,2016,72 (1): 106-119.), the obtained compound is confirmed to be Bacillomycin D (Bacillomycin D), and simultaneously the liquid phase peak area size is compared, and a strain with high yield of antibacterial lipopeptide (Bacillomycin D) is screened. The bacterial liquid of the strain after re-screening is preserved at-80 ℃.
4. Final screening results
Through the screening process, the strain with obvious inhibition effect on aspergillus flavus is finally obtained, and the strain has the performance of high-yield antibacterial lipopeptide (bacillomycin D). For convenience of recording and management, the inventors named it: strain BYA-KC-4.
The inhibition map of the active antibacterial lipopeptide produced by the strain BYA-KC-4 obtained by re-screening in the step 2.3 of the embodiment on the action of Aspergillus flavus is shown in figure 2, wherein the reference numeral 1 in the figure 2 is an ethanol control with a volume fraction of 95%, and the reference numeral 2 is an ethanol solution of the active antibacterial lipopeptide (wherein the solvent is ethanol with a volume fraction of 95%), and it can be seen from figure 2 that the prepared antibacterial lipopeptide has a significant inhibition effect on Aspergillus flavus.
FIG. 3 is a liquid chromatogram obtained by analyzing the secondary metabolite produced by the strain BYA-KC-4 in step 3 using a LC-MS analyzer, and according to the result of LC data analysis, it can be found that the strong absorption peaks a, b, and C within 16-20min are mainly C of Bacillamycin D (Bacillamycin D) differing by one subunit 14 、C 15 、C 16 Homologues having molecular weights of 1031.55, 1045.56 and 1059.57, respectively; as can also be seen from FIG. 3, other peaks include antibacterial lipopeptide components such as Bacilysin (Bacilysin), camellin (Fengycin), and Surfactin (Surfactin).
Furthermore, the inventor also carries out a genetic stability test of the bacterial strain BYA-KC-4 for producing the antibacterial lipopeptide, the test result is shown in figure 4, the inventor continuously subcultures the bacterial strain for 9 times, and the bacterial strain performance is still stable, and the bacteriostatic activity and the production capacity of the antibacterial lipopeptide (bacillomycin D) are still unchanged.
Example 2 morphological characteristics and molecular biological identification of Strain BYA-KC-4
The morphological characteristics are as follows:
diluting the bacterial liquid of the strain BYA-KC-4 obtained by re-screening by 100-1000 times, coating the diluted bacterial liquid on an LB (lysogeny broth) flat plate, and culturing for 24 hours at 37 ℃ to obtain bacterial colonies, wherein the bacterial colonies are shown in figure 5; the bacterial colony of the bacterial strain BYA-KC-4 is observed to be circular, smooth in surface, raised, opaque, milky white, neat in edge, certain in viscosity and l-2 cm in diameter; gram positive staining; 0.6-0.9 (width) multiplied by 1.0-1.5 μm (width), oval or oval, and no obvious expansion of the thalli after the spores are formed.
(II) physiological and biochemical characteristics:
the strain is positive in catalase reaction and VP reaction, and can reduce nitrate and hydrolyze starch and gelatin. The growth was normal in the liquid medium containing 1 to 7% by weight of NaCl, and the specific physiological and biochemical characteristics are shown in Table 2 below.
TABLE 2 measurement results of physiological and biochemical indicators
Note: + positive result: negative results are indicated.
(III) molecular biology identification:
and (3) performing PCR amplification on the cultured bacterial strain BYA-KC-4 bacterial liquid to obtain a 16S rDNA gene sequence, performing Blast comparison on NCBI, searching in a GenBank database, performing sequence homology analysis, and determining the bacterial strain BYA-KC-4 to be Bacillus subtilis (Bacillus subtilis) according to the sequencing result which is shown in a sequence SEQ ID NO. 1.
The bacillus subtilis BYA-KC-4 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation address is as follows: the microbial research institute of China academy of sciences No. 3, xilu No.1, beijing, chaoyang, has a preservation date of 2022 years, 9 months and 19 days, and a preservation number of CGMCC No.25758.
The sequencing result of the 16S rDNA of the strain is shown in SEQ ID NO.1, and specifically comprises the following steps:
1-60acgctggcgg cgtgcctaat acatgcaagt cgagcggaca gatgggagct tgctccctga
61-120tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc
121-180cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt
181-240ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg241-300ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag
301-360acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc
361-420tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg421-480gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg
481-540gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt541-600gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg
601-660gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg
661-720tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct
721-780gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc
781-840cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta841-900acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac
901-960gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac
961-1020caggtcttga catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga
1021-1080caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg
1081-1140agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt
1141-1200gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta
1201-1260cacacgtgct acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca
1261-1320caaatctgtt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct
1321-1360agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg
example 3 method for realizing high yield of antibacterial lipopeptide by using Bacillus subtilis BYA-KC-4
1. Culture of Bacillus subtilis BYA-KC-4
1.1 strain activation: taking out a glycerol freezing tube for storing the bacillus subtilis BYA-KC-4 from a refrigerator at the temperature of-80 ℃, freezing and thawing the glycerol freezing tube in ice bath, inoculating the thawed bacterial liquid into an LB solid slant culture medium by using an inoculating loop, culturing for 24 hours at the temperature of 30 ℃, and activating strains;
1.2 seed culture: after slant culture, inoculating a ring of slant lawn with an inoculating loop, inoculating into 10mL LB liquid medium (using a 50mL triangular shake flask), and culturing for 16-18 h at 30 ℃ and 180r/min in a shaking table to obtain a first-stage seed solution;
1.3 seed expanding culture: inoculating 1mL of the first-order seed liquid into 50mL of LB liquid culture medium (using a triangular shake flask with the volume of 250 mL), wherein the inoculation amount (volume fraction) is 2%, and culturing for 16-18 h at 30 ℃ and 180r/min of a shaking table to obtain a propagation seed liquid, wherein the concentration of the Bacillus subtilis BYA-KC-4 is 1x10 9 cfu/mL；
1.4 fermentation culture: inoculating 10mL of the expanded culture seed solution into 200mL of a fermentation medium (a triangular shake flask with the volume of 1L is used), wherein the inoculation amount (volume fraction) is 5%, and culturing for 48h at 30 ℃ and 180r/min of a shaking table to obtain 200mL of fermentation liquor, wherein the bacteria concentration in the fermentation liquor is about 10% (volume fraction).
2. Method for producing active antibacterial lipopeptide by using bacillus subtilis BYA-KC-4
2.1 centrifuging the bacillus subtilis fermentation liquor for 10min at the rotating speed of 8000r/min, removing thallus precipitates, combining and collecting fermentation supernatant;
2.2 taking 1L of fermentation supernatant, adjusting the pH of the supernatant to 2.5 by using a hydrochloric acid solution with the concentration of 6mol/L, wherein a precipitate is generated, and putting the supernatant in a refrigerator at 4 ℃ overnight to completely precipitate; pouring off part of the supernatant slightly the next day, centrifuging the rest suspension at 8000r/min for 10min, and collecting precipitate, wherein about 8-10 g of precipitate is contained in 1L of fermentation supernatant;
2.3 dissolving and extracting the obtained precipitate with 50mL of absolute ethanol repeatedly for 3 times, and mixing the extracting solutions; filtering and removing impurities from the ethanol extract with 0.22 μm microporous filter membrane (mixed membrane, mixed fiber microporous filter membrane water system/nylon filter membrane organic system, specification 50mm 0.22 μm, thickness 0.8 μm, purchased from Shanghai Xinya purification device factory) to obtain clarified extract (about 120-130 mL);
2.4 distilling the extract solution with rotary evaporator at 55 deg.C under reduced pressure until the ethanol solvent is completely evaporated; scraping off solid matters on the wall of the rotary steaming bottle and grinding the solid matters into powder to obtain an antibacterial lipopeptide extract, wherein the yield of the extract in each 1L of fermentation supernatant is 550-780 mg, and meanwhile, the content of the identified antibacterial lipopeptide in the extract is 96.57% by liquid chromatography peak area normalization calculation;
furthermore, the extract is separated and purified by adopting a macroporous resin and silica gel column chromatography method, and liquid phase and mass spectrum identification are carried out to obtain the antibacterial lipopeptide with the component of the bacillomycin D reaching more than 93 percent, specifically 93.36 percent, which shows that the method of the embodiment can realize the preparation of the antibacterial lipopeptide (bacillomycin D) with high yield.
Example 4 degradation of zearalenone toxin by active antibacterial lipopeptides obtained from the Bacillus subtilis BYA-KC-4
1. Sample treatment:
taking 0.7g of the active antibacterial lipopeptide prepared in the example 3, dissolving the active antibacterial lipopeptide in 6.3mL PBS (0.01 mol/L) buffer solution, centrifuging the solution for 10min under the condition of 10000r/min, and collecting supernatant as a sample solution; adding 300 μ L (2500 ppb concentration) of zearalenone toxin into 1200 μ L of sample solution as test group for reaction, and adding 300 μ L (2500 ppb concentration) of zearalenone toxin into 1200 μ L of PBS buffer as control group; the pH value of the reaction system is adjusted to 6.2, and the test group and the control group are respectively reacted at 37 ℃ for 2h, 5h, 12h, 24h, 48h and 72h, and 200 mu L of the samples are taken.
2. Detecting the content of zearalenone toxin (ELISA method, the following kit is an ELISA zearalenone toxin kit of Beijing Dubang, wherein the dosage of each reagent and the detection method refer to the specific using method in the kit):
2.1 taking out the kit from a refrigerator at 4 ℃ and placing the kit at room temperature for 30min; the corresponding microwells of the sample and standard were numbered on a 96-well plate, with 2 wells in parallel per well.
2.2 sucking 20 μ L of standard or sample and adding into corresponding micro-pores, respectively adding 100 μ L of working solution of enzyme conjugate, shaking gently, mixing, covering with cover plate, and reacting at 25 deg.C in dark environment for 15min.
2.3 carefully uncovering the cover plate film and drying the liquid in the holes; adding 250 mu L of deionized water into each hole, fully washing for 4-5 times at intervals of 10s each time, splashing the washing liquid in the plate holes, and patting the plate dry by using absorbent paper; if bubbles exist after the swatter is dried, the air bubbles are punctured by the sterilized gun head.
2.4 adding 50 mu L of substrate A liquid into each hole, adding 50 mu L of substrate B liquid, lightly shaking and uniformly mixing, and reacting for 5min in a dark place at 25 ℃; after the reaction, blue with different depths can be seen, the darker the color is, the smaller the concentration of the representative toxin is, and if the whole color is lighter, the reaction time can be prolonged to 7min.
2.5 adding 50 μ L stop solution into each well, shaking gently and mixing well, and stopping reaction.
2.6 the absorbance of each well was measured at 450nm using a microplate reader.
3. And (3) preparing a standard curve:
preparing zearalenone toxin standard solution with the concentration of 0ppb, 20ppb, 80ppb, 240ppb and 1000ppb respectively; average value of absorbance values (E) of each concentration of zearalenone toxin standard solution and sample obtained Red blood in the stomach ) Divided by the absorbance value (E) of the first standard (0 standard) 0 red ) Multiplying by 100 percent, namely a percent absorbance value;
percent absorbance value (%) = E Red blood in the stomach /E 0 red ×100％；
Drawing a standard curve by taking the percent absorbance of the standard substance as a vertical coordinate and taking the logarithm of the concentration (ppb) of the zearalenone toxin standard substance as a horizontal coordinate to obtain a regression equation; and substituting the percent absorbance of the sample into an equation to obtain the zearalenone toxin concentration in the sample.
4. The degradation rate calculation formula is as follows:
zearalenone toxin degradation rate (%) = (a) 0 -A)/A 0 *100；
A 0 (ppb) mean zearalenone toxin concentration for the control group;
a (ppb) corresponds to the mean concentration of zearalenone toxin per hour;
5. and (3) detection results:
as shown in FIG. 6, it can be seen that when zearalenone toxin is degraded by using the active antibacterial lipopeptide produced by Bacillus subtilis BYA-KC-4 of the present invention, the degradation effect increases with time; reacting for 2 hours, wherein the degradation rate of the zearalenone toxin by the active antibacterial lipopeptide of the bacillus subtilis BYA-KC-4 is 27%; reacting for 5 hours, wherein the degradation rate of zearalenone toxin is 58%; the reaction lasts for 12 hours, and the degradation rate of zearalenone toxin is 77%; the reaction lasts for 24 hours, and the degradation rate of zearalenone toxin is 84%; reacting for 48 hours, wherein the degradation rate of zearalenone toxin is 90%; the reaction time is 72 hours, and the degradation rate of zearalenone toxin is 92%.
Example 5 degradation of emetic toxin by active antimicrobial lipopeptides obtained using the Bacillus subtilis BYA-KC-4
1. Sample treatment:
taking 0.7g of the active antibacterial lipopeptide prepared in the example 3, dissolving the active antibacterial lipopeptide in 6.3mL of PBS (0.01 mol/L) buffer solution, centrifuging the solution for 10min under the condition of 10000r/min, and collecting the supernatant as a sample solution; adding 300 mu L (concentration 10000 ppb) of vomitoxin into 1200 mu L of sample solution to be used as a test group for reaction, and adding 300 mu L (concentration 10000 ppb) of vomitoxin into 1200 mu L of PBS buffer solution as a control group; the pH value of the reaction system is adjusted to 6.2, and the test group and the control group are respectively reacted at 37 ℃ for 2h, 5h, 12h, 24h, 48h and 72h, and 200 mu L of the sample is taken.
2. Detecting the content of vomitoxin (ELISA method, the following kit is ELISA vomitoxin kit of Beijing Dubang, wherein the dosage and detection method of each reagent refer to the specific using method in the kit):
2.1 taking out the kit from a refrigerator at 4 ℃ and placing the kit at room temperature for 30min; the corresponding microwells of the sample and standard were numbered on a 96-well plate, with 2 wells in parallel per well.
2.2 sucking 20 μ L of standard substance or sample, adding into corresponding micropores, adding 100 μ L of enzyme conjugate working solution, shaking gently, mixing, covering with cover plate, and reacting at 25 deg.C in dark environment for 15min.
2.3 carefully uncovering the cover plate film and spin-drying liquid in the holes; adding 250 mu L of deionized water into each hole, fully washing for 4-5 times at intervals of 10s each time, splashing the washing liquid in the plate holes, and patting the plate dry by using absorbent paper; if bubbles exist after the swatting is dry, the bubbles are punctured by a sterilized gun head.
2.4 adding 50 mu L of substrate A liquid into each hole, adding 50 mu L of substrate B liquid, lightly shaking and uniformly mixing, and reacting for 5min in a dark place at 25 ℃; after the reaction, blue with different depths can be seen, the darker the color is, the smaller the concentration of the representative toxin is, and if the whole color is lighter, the reaction time can be prolonged to 7min.
2.5 adding 50 μ L stop solution into each well, shaking gently and mixing well, and stopping reaction.
2.6 the absorbance of each well was measured at 450nm using a microplate reader.
3. And (3) preparing a standard curve:
vomitoxin standard solutions were prepared at concentrations of 0ppb, 150ppb, 500ppb, 1500ppb, and 5000ppb, respectively. Average value of absorbance values of obtained vomitoxin per concentration standard solution and sample (E) Vomiting (vomiting) ) Divided by the absorbance value (E) of the first standard (0 standard) 0 vomiting ) Then multiplying by 100 percent, namely the percent absorbance value;
percent absorbance value (%) = E Vomiting (vomiting) /E 0 vomiting ×100％；
Drawing a standard curve by taking the percent absorbance of the standard substance as a vertical coordinate and the logarithm of the concentration (ppb) of the vomitoxin standard substance as a horizontal coordinate to obtain a regression equation; substituting the percent absorbance of the sample into an equation to obtain the concentration of the vomitoxin in the sample.
4. The degradation rate calculation formula is as follows:
vomitoxin degradation rate (%) = (B) 0 -B)/B 0 *100；
B 0 (ppb) mean vomitoxin concentration for control group;
b (ppb) corresponding to the mean hourly vomitoxin concentration;
5. and (3) detection results:
as shown in FIG. 7, it can be seen that when the active antibacterial lipopeptide produced by the Bacillus subtilis BYA-KC-4 of the present invention is used for degrading vomitoxin, the degradation effect is increased along with the change of time; reacting for 2 hours, wherein the degradation rate of the active antibacterial lipopeptide of the bacillus subtilis BYA-KC-4 to vomitoxin is 23%; the reaction lasts for 5 hours, and the degradation rate of the vomitoxin is 34 percent; reacting for 12 hours, wherein the degradation rate of the vomitoxin is 55%; reacting for 24 hours, wherein the degradation rate of the vomitoxin is 61%; reacting for 48 hours, wherein the degradation rate of the vomitoxin is 67%; after the reaction is carried out for 72 hours, the degradation rate of the vomitoxin is 81 percent.
Example 6 degradation of Aflatoxin B Using the active antimicrobial lipopeptide obtained from Bacillus subtilis BYA-KC-4 1
1. Sample treatment:
taking 0.7g of the active antibacterial lipopeptide prepared in the example 3, dissolving the active antibacterial lipopeptide in 6.3mL of PBS (0.01 mol/L) buffer solution, centrifuging the solution for 10min under the condition of 10000r/min, and collecting the supernatant as a sample solution; a sample solution of 1200. Mu.L was added to 300. Mu.L (200 ppb concentration) of aflatoxin B 1 Performing the reaction as a test group; the control group was 1200. Mu.L of PBS buffer to which 300. Mu.L (200 ppb concentration) of aflatoxin B was added 1 (ii) a The pH value of the reaction system is adjusted to 6.2, and the test group and the control group are respectively reacted at 37 ℃ for 2h, 5h, 12h, 24h, 48h and 72h, and 200 mu L of the sample is taken.
2. Aflatoxin B 1 Content detection (ELISA method, the following kit is ELISA aflatoxin B of Shenzhen Yirui biotechnology 1 The kit comprises the following specific application methods for the dosage and detection method of each reagent:
2.1 taking out the kit from a refrigerator at 4 ℃ and placing the kit at room temperature for 30min; the corresponding microwells of the sample and standard were numbered on a 96-well plate, with 2 wells in parallel per well.
2.2 sucking 20 μ L of standard substance or sample, adding into corresponding micropores, adding 100 μ L of enzyme conjugate working solution, shaking gently, mixing, covering with cover plate, and reacting at 25 deg.C in dark environment for 15min.
2.3 carefully uncovering the cover plate film and spin-drying liquid in the holes; adding 250 mu L of deionized water into each hole, fully washing for 4-5 times at intervals of 10s each time, splashing the washing liquid in the plate holes, and patting the plate dry by using absorbent paper; if bubbles exist after the swatter is dried, the air bubbles are punctured by the sterilized gun head.
2.4 adding 50 mul of substrate A liquid into each hole, adding 50 mul of substrate B liquid, lightly shaking and uniformly mixing, and reacting for 5min in a dark place at 25 ℃; after the reaction, blue with different depths can be seen, the darker the color is, the smaller the concentration of the representative toxin is, and if the overall color is lighter, the reaction time can be prolonged to 7min.
2.5 adding 50 μ L stop solution into each well, shaking gently and mixing well, and stopping reaction.
2.6 the absorbance of each well was measured at 450nm using a microplate reader.
3. Preparation of a standard curve:
preparing aflatoxin B 1 The concentrations of the standard solutions were 0ppb, 2ppb, 5ppb, 20ppb, and 50ppb, respectively. The obtained aflatoxin B 1 Average value of absorbance values of each concentration standard solution and sample (E) Yellow colour ) Divided by the absorbance value (E) of the first standard (0 standard) 0 yellow ) Then multiplying by 100 percent, namely the percent absorbance value;
percent absorbance value (%) = E Yellow colour /E 0 yellow ×100％；
Taking the percent absorbance of the standard as the ordinate and the aflatoxin B 1 Taking the logarithm of the concentration (ppb) of the standard substance as a horizontal coordinate, drawing a standard curve, and obtaining a regression equation; substituting the percent absorbance of the sample into an equation to obtain aflatoxin B in the sample 1 And (4) concentration.
4. The degradation rate calculation formula is as follows:
aflatoxin B 1 Degradation rate (%) = (C) 0 -C)/C0*100；
C 0 (ppb) control Aflatoxin B 1 Average concentration value;
c (ppb) corresponding to aflatoxin B per hour 1 Average concentration value;
5. and (3) detection results:
as shown in FIG. 8, it can be seen that the active antibacterial lipopeptide produced by the Bacillus subtilis BYA-KC-4 of the present invention can be used for aflatoxin B 1 When degradation is carried out, the degradation effect is increased along with the change of time; after 2h of reaction, the active antibacterial lipopeptide of the bacillus subtilis BYA-KC-4 has effect on aflatoxin B 1 The degradation rate of (2) is 25%; reacting for 5h to aflatoxin B 1 The degradation rate of (2) is 31%; reacting for 12h, and reacting on aflatoxin B 1 The degradation rate of (2) is 45%; reacting for 24h, and reacting on aflatoxin B 1 The degradation rate of (2) is 60%; reacting for 48h, and reacting on aflatoxin B 1 The degradation rate of (2) is 79%; reacting for 72 hours to react with aflatoxin B 1 The degradation rate of (2) was 84%.
In conclusion, the screened bacillus subtilis BYA-KC-4 has the capability of efficiently degrading various mycotoxins and the performance of high-yield active antibacterial lipopeptide through experimental verification, and therefore, the bacillus subtilis has good application value in the aspects of developing novel biological detoxifiers, feed additives and the like, reducing the pollution of mycotoxins to feeds or processing raw materials and the like.
The foregoing examples are illustrative of embodiments of the present invention, and although the present invention has been illustrated and described with reference to specific examples, it should be appreciated that embodiments of the present invention are not limited by the examples, and that various changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Sequence listing
SEQ ID NO.1 shows the 16S rDNA sequencing result of the strain
acgctggcggcgtgcctaatacatgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtg
ggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcagacataaaaggtggctt
cggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagaggg
tgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggag
caacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttcaaatagggcggcaccttgacggta
cctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctc
gcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagt
ggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagc
gaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgct
gcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagc
atgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtg
acaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagtt
gggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgc
tacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgc
gtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccg。
Claims (10)
1. An antibacterial lipopeptide high-producing strain is characterized in that the strain is Bacillus subtilis (Bacillus subtilis), and the strain is named as follows by the inventor: the bacillus subtilis BYA-KC-4 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and the preservation addresses are as follows: the microbiological research institute of the institute of sciences of china, no. 3, west way, no.1, north chen, chaoyang district, the preservation date: 2022, 9/19, accession no: CGMCC No.25758.
2. Use of the bacillus subtilis of claim 1 for the preparation of an antimicrobial lipopeptide by a biological fermentation process.
3. A method for producing antibacterial lipopeptide by using the bacillus subtilis through fermentation is characterized by comprising the following steps:
(1) Activating strains: inoculating bacillus subtilis into an LB solid culture medium, and culturing at 28-32 ℃ for 12-24 h to obtain an activated strain;
(2) Seed culture: inoculating the activated strain to an LB liquid culture medium, and culturing at 28-32 ℃ for 16-18 h to obtain a first-stage seed solution;
(3) And (3) amplification culture: taking the first-stage seed liquid to inoculate to an LB liquid culture medium, and culturing for 16-18 h at 28-32 ℃ to obtain a culture expanding seed liquid;
(4) Fermentation culture: inoculating the expanded culture seed liquid to a fermentation culture medium, and culturing at 28-32 ℃ for 12-48 h to obtain a fermentation liquid;
(5) Centrifuging the fermentation liquor obtained in the step (4) to obtain fermentation supernatant, adjusting the pH of the fermentation supernatant to 2.5 by using acid liquor to generate precipitate, standing for 2-24 hours to completely precipitate, removing part of supernatant to obtain suspension, and centrifuging to obtain precipitate;
(6) And (3) extracting the precipitate obtained in the step (5) by using absolute ethyl alcohol to obtain an extracting solution, filtering to remove impurities, and distilling the ethanol solvent under reduced pressure at the temperature of 50-60 ℃ to obtain a solid product, namely the antibacterial lipopeptide.
4. The method according to claim 3, wherein the seed liquid obtained in step (3) has a concentration of 1x10 9 cfu/mL～2x10 9 cfu/mL。
5. The method of claim 3, wherein the amount of inoculum for the fermentation culture in step (4) is 2-5%.
6. Use of the bacillus subtilis of claim 1 for degrading mycotoxins.
7. Use of an antimicrobial lipopeptide prepared by the method of claim 3 for the degradation of mycotoxins.
8. The use of claim 7, wherein said antimicrobial lipopeptide is capable of simultaneously degrading a plurality of mycotoxins, said mycotoxins including zearalenoneKetotoxin, vomitoxin, aflatoxin B 1 。
9. A method of degrading mycotoxins using the antimicrobial lipopeptide prepared according to claim 3, comprising the steps of:
diluting the prepared antibacterial lipopeptide, centrifuging to obtain supernatant, mixing the supernatant with a mycotoxin solution for reaction, adjusting a reaction system to be acidic, and reacting for 2-72 h at 35-38 ℃.
10. The method of claim 9, wherein the mycotoxin solution is an aqueous zearalenone solution, an aqueous vomitoxin solution, and aflatoxin B 1 One, two or three of the aqueous solutions;
the concentration of the supernatant is 50-100 mug/mL;
the concentration of the mycotoxin solution is 200-10000 ppb;
the volume ratio of the supernatant to the mycotoxin solution is (3-12): 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310082062.0A CN115948305B (en) | 2023-01-16 | 2023-01-16 | Bacillus subtilis capable of efficiently degrading various mycotoxins and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310082062.0A CN115948305B (en) | 2023-01-16 | 2023-01-16 | Bacillus subtilis capable of efficiently degrading various mycotoxins and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115948305A true CN115948305A (en) | 2023-04-11 |
| CN115948305B CN115948305B (en) | 2023-08-15 |
Family
ID=87291264
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310082062.0A Active CN115948305B (en) | 2023-01-16 | 2023-01-16 | Bacillus subtilis capable of efficiently degrading various mycotoxins and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115948305B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6183736B1 (en) * | 1998-04-07 | 2001-02-06 | Usda/Ars Southern Regional Research Center | Small peptides with antipathogenic activity, treated plants and methods for treating same |
| CN110272854A (en) * | 2019-07-29 | 2019-09-24 | 北京林业大学 | A kind of bacillus subtilis strain and its application |
| CN111440744A (en) * | 2020-04-17 | 2020-07-24 | 内蒙古中能生物科技有限公司 | Test method for degrading feed mycotoxin by using bacillus subtilis |
| CN114703096A (en) * | 2022-04-01 | 2022-07-05 | 天津科技大学 | Bacillus belgii and fermented feed degrading microbial toxin and application thereof |
| CN115305213A (en) * | 2021-11-02 | 2022-11-08 | 河北农业大学 | Bacillus subtilis and culture method and application thereof |
-
2023
- 2023-01-16 CN CN202310082062.0A patent/CN115948305B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6183736B1 (en) * | 1998-04-07 | 2001-02-06 | Usda/Ars Southern Regional Research Center | Small peptides with antipathogenic activity, treated plants and methods for treating same |
| CN110272854A (en) * | 2019-07-29 | 2019-09-24 | 北京林业大学 | A kind of bacillus subtilis strain and its application |
| CN111440744A (en) * | 2020-04-17 | 2020-07-24 | 内蒙古中能生物科技有限公司 | Test method for degrading feed mycotoxin by using bacillus subtilis |
| CN115305213A (en) * | 2021-11-02 | 2022-11-08 | 河北农业大学 | Bacillus subtilis and culture method and application thereof |
| CN114703096A (en) * | 2022-04-01 | 2022-07-05 | 天津科技大学 | Bacillus belgii and fermented feed degrading microbial toxin and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| BERTUZZI T等: "Reduction of Mycotoxigenic Fungi Growth and Their Mycotoxin Production by Bacillus subtilis QST 713", 《TOXINS (BASEL)》, vol. 14, no. 11, pages 1 - 10 * |
| JI C等: "Review on biological degradation of mycotoxins", 《ANIM NUTR》, vol. 02, no. 03, pages 127 - 133 * |
| 刘佳欣等: "枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定", 《工业微生物》, vol. 47, no. 02, pages 11 - 17 * |
| 常艺海等: "一株产抗菌肽芽孢杆菌的筛选、鉴定及其发酵条件优化研究", 《饲料研究》, vol. 45, no. 20, pages 65 - 69 * |
| 田玉虎等: "霉菌毒素降解菌株和吸附剂的体外效果评估和筛选", 《饲料研究》, vol. 43, no. 05, pages 61 - 64 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115948305B (en) | 2023-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105385616B (en) | The bacillus subtilis of efficient degradation zearalenone and its application | |
| CN105087444B (en) | The bacillus amyloliquefaciens of degrading zearalenone and its application | |
| KR101280811B1 (en) | Microorganism having zearalenone decomposing activity, method for degrading zearalenone using thereof and feed additives comprising the same | |
| CN110564640B (en) | Siamese bacillus WF2019 strain for degrading aflatoxin B1 and application thereof | |
| CN114134075B (en) | Bacillus bailii capable of producing complex enzyme with high yield and simultaneously degrading mycotoxin with high efficiency and application thereof | |
| CN107201322A (en) | Bacillus subtilis and its application for degrading aflatoxin B 1 | |
| CN109182197B (en) | Bacillus amyloliquefaciens CPLK1314 and application thereof in feed storage | |
| CN103387950A (en) | Devosia sp and application thereof in degrading vomitoxin | |
| CN110042072B (en) | Aflatoxin B degradation1And application thereof | |
| CN111778178B (en) | Application of marine streptomyces griseoflavus HN60 in antibacterial aspect | |
| CN116855414B (en) | Bacillus belicus and application thereof in fermented bean products | |
| CN114672440B (en) | Bacillus subtilis BS273 and application thereof | |
| CN111394270B (en) | Nocardia gamboge and application thereof | |
| CN115948305B (en) | Bacillus subtilis capable of efficiently degrading various mycotoxins and application thereof | |
| CN113999791B (en) | Pseudo-intermediate Brucella for adsorbing vomitoxin and aflatoxin B1 and application thereof | |
| CN114517170A (en) | Bacillus subtilis for degrading vomitoxin and application thereof | |
| CN109161500B (en) | Siamese bacillus ZJ-2018-1, mycotoxin degrading microbial inoculum and application | |
| CN113897319A (en) | Bacillus amyloliquefaciens and application thereof | |
| CN111011707A (en) | Preparation method of flavor detoxified corn steep liquor | |
| CN116396883B (en) | Bacillus subtilis capable of degrading zearalenone and vomitoxin at high temperature and high efficiency and microbial inoculum thereof | |
| CN115044502B (en) | Disease-inhibiting growth-promoting bacillus cereus YT2-1C and application thereof | |
| CN113755410B (en) | Bacillus licheniformis for degrading vomitoxin and application thereof | |
| CN115404184B (en) | Alkaline halophiles clausii PA21 and application thereof in degradation of potato solanine | |
| CN116445323A (en) | Bacillus bailii capable of antagonizing growth of toxigenic mold and degrading vomitoxin and application thereof | |
| CN114698767B (en) | Biological agent for removing mycotoxin in feed as well as preparation method and application of biological agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |