CN115948305B - 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
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Abstract
The invention belongs to the technical field of microbial preparations and natural product application, and particularly relates to bacillus subtilis capable of efficiently degrading various mycotoxins and application thereof. The invention provides an active antibacterial lipopeptide high-yield strain, which is bacillus subtilis. The invention also provides application of the bacillus subtilis in preparation of the antibacterial lipopeptid, and the bacillus subtilis is used for preparing and producing the antibacterial lipopeptid 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 feeding strain only acts on one mycotoxin at the present stage and is difficult to be practically applied due to the limited application range is solved. The strain can be used for developing novel biological detoxication agents, feed additives and the like, is very suitable for being used as the feed additives, and has good application value and market popularization prospect in feed production and cultivation industries.
Description
Technical Field
The invention belongs to the technical field of microbial preparations and natural product application, and particularly relates to bacillus subtilis for efficiently degrading various mycotoxins and application thereof, in particular to bacillus subtilis with high yield of active antibacterial lipopeptid and application of the high-yield strain and the antibacterial lipopeptid in degrading mycotoxins.
Background
The quality safety of the feed is the first gateway for guaranteeing the safety of cultured products and food safety, and preventing the feed from mildew becomes a common concern of the feed industry. At least 25% of grains in the world are currently contaminated with mycotoxins, estimated by the united nations grain and agriculture organization (FAO). The pollution condition of the mycotoxin in China is more optimistic, and the pollution rate of the mycotoxin is up to 90 percent. Among the various mycotoxins, the major hazards are aflatoxins, zearalenone (F2 toxin), and vomitoxin and trichothecene (T2 toxin).
The detection report of the mycotoxins of the feed and the raw materials in China in 2021 is provided by the Shandong Longchang animal protection research center, 1088 feed samples in various places in China are detected, and the coexistence phenomenon of various toxins in all samples is obvious, wherein the samples for detecting 1 mycotoxin account for 5%, the samples for detecting 2 mycotoxins account for 20%, and the samples for detecting 3 or more mycotoxins account for 75%. Aflatoxin B in different types of samples 1 The positive detection rate of the zearalenone and vomitoxin is more than 87%.
Mycotoxins are toxic secondary metabolites produced by fungi (moulds). Aflatoxin is produced by aspergillus flavus, and can cause liver necrosis, reduce production efficiency, reduce milk yield, cause congenital defects, tumor and inhibit immune system. Zearalenone toxin and vomitoxin are both produced by fusarium, have various strong toxicities, and are extremely harmful to animals and humans. Zearalenone and its metabolites can cause oestrogenic and reproductive disorders in a wide variety of female animals, for example feeding on contaminated zearalenone can cause oestrogenic toxicity in sows, resulting in a range of reproductive disorders such as pseudopregnancy, infertility, ovarian malformation and abortion. Vomitoxin can have an effect on the digestive system, blood system, central system, and calcium-phosphorus metabolism of animals. The low dose of vomitoxin in the feed can cause symptoms such as appetite reduction, weight loss, metabolic disorder and the like of animals, and the high dose can cause the animals to vomit, so that pigs are the animals most sensitive to the vomitoxin.
A great deal of research shows that mycotoxins are widely used in grains, animal feeds and agricultural and sideline products, and especially the pollution of zearalenone, aflatoxin and vomitoxin in processed foods or produced feeds with corn, wheat, rice, peanut and the like as raw materials is the most serious. Although some processes (e.g., high temperature tempering) may kill some microorganisms such as mold, toxins produced by mold metabolism are difficult to eliminate. After the mycotoxin pollutes the feed, the animal health and the human health are directly endangered, and the application of the grain in food medicine, feed and deep processing industry is limited. 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 high.
In recent years, the adoption of microorganism probiotics and the generated active substances as feed additives plays a key role in mould prevention of feeds. However, the probiotic feed additive used at present mainly only has degradation effect on single mycotoxin, for example, a strain of zearalenone toxin degrading saccharomycete and application thereof (CN 105794963A), a strain of bacillus subtilis for degrading aflatoxin (CN 101705203B), an strain of enterococcus faecium GXSCU1 for efficiently degrading vomitoxin, a microbial agent, and preparation methods and application thereof (CN 114437983A) of the probiotic feed additive.
Feed is often affected by various mycotoxins due to the influence of raw material sources and storage environment, and until now, it has not been found that one microorganism can simultaneously and irreversibly degrade various mycotoxins. According to the invention, the bacillus subtilis is obtained through screening, and the secondary metabolite is extracted and analyzed, so that the bacillus subtilis and the active antibacterial lipopeptid secreted by the bacillus subtilis can simultaneously and efficiently degrade at least three mycotoxins, and the bacillus subtilis has the advantages of high detoxification activity, strong specificity, mild effect and wide application prospect in the field of feed additives.
Disclosure of Invention
The mycotoxin has wide range and large harm because of polluting grains and feeds, and the problem of low degradation efficiency when the single use of the microorganism capable of degrading the mycotoxin is taken as a microbial agent or a feed additive is prepared at the present stage, so the method is not suitable for actual production. Therefore, the invention provides a beneficial microorganism (bacillus subtilis) aiming at the defects existing in the prior art, the microorganism and the active metabolite (antibacterial lipopeptide) secreted by the microorganism can efficiently degrade various mycotoxins in feed or processing raw materials, especially can reduce the toxicity of aflatoxin, zearalenone and vomitoxin to human or animals, improves the nutritive value of the feed or food processing raw materials, and lays a foundation for developing a microecological preparation which is efficient, safe and environment-friendly and can degrade mycotoxins.
Furthermore, the invention also aims to provide application of the bacillus subtilis in preparation of antibacterial lipopeptides.
Furthermore, the invention also aims to provide application of the bacillus subtilis or the antibacterial lipopeptid in degrading various mycotoxins.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
an active antibacterial lipopeptide high-yield strain, wherein the strain is bacillus subtilis (Bacillus subtilis) and is obtained by screening and separating deep sea mud of a large continuous long sea cucumber deep sea culture base; for ease 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 round, has smooth surface, is raised, is opaque, is milky and has neat edge, certain viscosity and diameter l-2 cm; gram positive staining; spores are 0.6-0.9 (width). Times.1.0-1.5 μm (width), oval or oval, and the bacterial cells do not obviously expand after the spores are formed.
The strain has been deposited by the inventors at 2022, 9 and 19 days, with the deposit address: the China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, national academy of sciences of China, no. 3, north Chen West Lu, 1, of the Chao, beijing, and the strain collection number is: CGMCC No.25758.
Furthermore, the invention also provides application of the bacillus subtilis in preparation of the antibacterial lipopeptid, and the bacillus subtilis is used for preparing the antibacterial lipopeptid by a biological fermentation method.
Further, the invention also provides a method for producing the antibacterial lipopeptide by utilizing the bacillus subtilis fermentation, which comprises the following steps:
(1) Activating strains: inoculating bacillus subtilis into LB solid medium, culturing for 12-24 h at 28-32 ℃ to obtain activated strain;
(2) Seed culture: inoculating the activated strain to an LB liquid culture medium, and culturing for 16-18 h at 28-32 ℃ to obtain primary seed liquid;
(3) And (3) performing expansion culture: inoculating the first-stage seed liquid into an LB liquid culture medium, and culturing for 16-18 h at 28-32 ℃ to obtain a seed liquid for expanding culture;
(4) Fermentation culture: inoculating the seed liquid to a fermentation medium, and culturing for 12-48 h at 28-32 ℃ to obtain fermentation liquid;
(5) Centrifuging the fermentation liquor obtained in the step (4) to obtain fermentation supernatant, regulating 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 the supernatant to obtain suspension, and centrifuging to obtain precipitate;
(6) Extracting the precipitate obtained in the step (5) with absolute ethyl alcohol to obtain an extracting solution, filtering to remove impurities, and distilling the ethanol solvent under reduced pressure at 50-60 ℃ to obtain a solid product, namely the antibacterial lipopeptide.
Specifically, the composition of the LB liquid medium is as follows: 10.0g of tryptone, 5.0g of yeast extract and 10.0g of NaCl in each liter of culture medium; when in use, the pH is regulated to 7.0-7.2, and the sterilization is carried out 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 10.0g of tryptone, 5.0g of yeast extract, 10.0g of NaCl and 18.0g of agar powder are added in each liter of culture medium; when in use, the pH is regulated to 7.0-7.2, and the sterilization is carried out for 20min at 121 ℃.
Specifically, the composition of the fermentation medium is as follows: glucose 10.0 g/liter of medium, bovine bone peptone 10.0g,NaCl 5.0g,K 2 HPO4 0.5g，MgSO 4 0.1g，MnSO 4 0.01g，FeSO 4 0.001g; the pH was adjusted to 7.0 when used, and sterilized at 121deg.C for 20min.
Specifically, the inoculum size (volume fraction) of the expansion culture in the step (3) is 1-2%.
Specifically, the seed liquid obtained in the step (3) has a concentration of 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 liquor in the step (5) is hydrochloric acid solution with the concentration of 5-8 mol/L.
Specifically, the solid-to-liquid ratio of the precipitate to the absolute ethanol in the step (6) is 1g:20mL.
Furthermore, based on a general inventive concept, the invention also provides application of the bacillus subtilis in degradation of mycotoxin.
Furthermore, based on a general inventive concept, the invention also provides application of the antibacterial lipopeptid in degradation of mycotoxin.
In particular, the antimicrobial lipopeptides are capable of simultaneously degrading a variety of mycotoxins including, but not limited to, zearalenone toxin, vomitoxin, aflatoxin B 1 。
Further, based on a general inventive concept, the present invention also provides a method for degrading mycotoxins using the antibacterial lipopeptides, comprising the steps of:
diluting the prepared antibacterial lipopeptide, centrifuging to obtain supernatant, mixing the supernatant with a mycotoxin solution for reaction, regulating the reaction system to be acidic, and reacting for 2-72 h at 35-38 ℃.
Specifically, the pH value of the reaction system is regulated to 6.2 during the reaction.
Specifically, the mycotoxin is zearalenone toxin, vomitoxin and/or aflatoxin B 1 。
Specifically, the mycotoxin solution is zearalenone toxic water solution, vomitoxin water 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 4:1.
Further preferably, the volume of the supernatant is 1000 to 1200. Mu.L, and the volume of the mycotoxin solution is 100 to 300. Mu.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 a mycotoxin degrading feed additive.
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 of the bacterial powder added into each kilogram of feed.
Further, the invention also provides a bacterial agent for degrading mycotoxin, the active ingredients of the bacterial agent comprise bacillus subtilis BYA-KC-4 or a mutant strain 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。
Furthermore, the invention also provides a preparation method of the mycotoxin degrading bacterial agent, which comprises the following steps:
1) Activating strains: inoculating bacillus subtilis into LB solid medium, culturing for 12-24 h at 28-32 ℃ to obtain activated strain;
2) Seed culture: inoculating the activated strain to an LB liquid culture medium, and culturing for 16-18 h at 28-32 ℃ to obtain primary seed liquid;
3) And (3) performing expansion culture: inoculating the first-stage seed liquid into an LB liquid culture medium, and culturing for 16-18 h at 28-32 ℃ to obtain a seed liquid for expanding culture;
4) Fermentation culture: inoculating the seed liquid to fermentation medium, and culturing at 28-32 deg.c for 12-48 hr to obtain fermented liquid as liquid microbial inoculum.
Specifically, the inoculum size (volume fraction) of the expansion culture in step 3) is 1-2%.
Specifically, the seed liquid obtained in step 3) has a concentration of 1×10 9 cfu/mL～2x10 9 cfu/mL。
Specifically, the inoculum size (volume fraction) of the fermentation culture in step 4) is 2-5%.
Further 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 3:3:2:2.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention obtains a strain with obvious inhibition effect on aspergillus flavus through repeated screening, and the strain can secrete active antibacterial lipopeptide with higher yield; by means of liquid analysis, it can be obtained that the main component of the antibacterial lipopeptide secreted by the strain is bacitracin D (Bacillomycin D). Studies have shown that bacitracin D is the only secondary metabolite obtained from microorganisms and identified as resistant to Aspergillus flavus and various molds, as well as mycotoxins, and therefore the strains screened in accordance with the present invention can be used to degrade various mycotoxins.
2. Experiments prove that the active antibacterial lipopeptide secreted by the bacillus subtilis BYA-KC-4 obtained by screening has a degradation rate of more than 90 percent on zearalenone toxin, a degradation rate of more than 80 percent on vomit toxin and an aflatoxin B 1 The degradation rate of the composite is more than 80 percent, and the composite has the capability of efficiently degrading various mycotoxins.
3. The bacillus subtilis BYA-KC-4 can secrete various enzymes such as lipase, amylase and protease, has high enzyme activity, is very beneficial to the health of farmed animals, is suitable for being used as an animal feed additive or a microbial inoculum, promotes the growth and development of animals, and improves the utilization rate of feed.
4. The bacillus subtilis BYA-KC-4 also has good stress resistance, strong acid and strong alkali resistance, 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 for being used as a feed additive, can efficiently degrade various mycotoxins, can secrete various digestive enzymes, has strong stress resistance, and is nontoxic and harmless to animals. Therefore, the strain is used for developing novel biological detoxication agents, feed additives and the like, reduces the pollution of feed or processing raw materials to mycotoxin, improves the feed utilization rate and the nutritive value, and has good application value and market popularization prospect in feed production and cultivation industries.
Drawings
FIG. 1 shows the results of the rescreening of example 1;
FIG. 2 is a diagram showing the antibacterial effect of the active antibacterial lipopeptid produced by the strain BYA-KC-4 obtained by re-screening in example 1 on Aspergillus flavus; wherein, the reference numeral 1 is ethanol contrast, the reference numeral 2 is ethanol solution of active antibacterial lipopeptide;
FIG. 3 is a liquid chromatogram of the analysis of secondary metabolites produced by strain BYA-KC-4 using a LC-MS analyzer in example 1;
FIG. 4 is a graph showing the genetic stability of strain BYA-KC-4 passage 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 active antimicrobial lipopeptides produced by Bacillus subtilis BYA-KC-4 in example 4 of the present invention;
FIG. 7 is a graph showing the time course of degradation of vomitoxin by active antimicrobial lipopeptides produced by Bacillus subtilis BYA-KC-4 in example 5 of the present invention;
FIG. 8 shows degradation of aflatoxin B by active antibacterial lipopeptides produced by Bacillus subtilis BYA-KC-4 in example 6 of the present invention 1 A time-dependent profile.
Detailed Description
For a better understanding of the present invention, the present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples, and all changes and equivalents that do not depart from the spirit of the present invention are intended to be 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 adding deionized water to 1.0L; sterilizing at 121 deg.c and pH 7.0-7.2 for 20min.
LB liquid medium: 10.0g of tryptone, 5.0g of yeast extract, 10.0g of NaCl and adding deionized water to 1.0L; sterilizing at 121 deg.c and pH 7.0-7.2 for 20min.
PDA medium: 200.0g of potato, 20.0g of glucose and 20.0g of agar, and adding deionized water to 1.0L; natural pH, sterilizing at 115 deg.C for 30min.
Fermentation medium: glucose 10.0g, bovine bone peptone 10.0g,NaCl 5.0g,K 2 HPO4 0.5g，MgSO 4 0.1g，MnSO 4 0.01g，FeSO 4 0.001g, deionized water was added to 1.0L; and sterilizing at 121 ℃ for 20min at pH 7.0.
Preparation of sterile physiological saline: 8.5g of NaCl is dissolved in a small amount of distilled water, and then distilled water is added to 1000mL; sterilizing at 121deg.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, dissolving in 800mL distilled water, adjusting the pH value of the solution to 7.5 by NaOH, and finally adding distilled water to a constant volume of 1L; sterilizing at 121deg.C for 20min.
Example 1 screening method of strains
Example 1 provides a screening method for bacillus subtilis with mycotoxin resistance, which comprises the following specific steps:
1. isolation of strains:
taking 1g of sea mud sample from deep sea mud of a large continuous sea cucumber deep sea culture base, adding 10mL of sterile physiological saline, fully vibrating and uniformly mixing to obtain sample liquid, and carrying out gradient dilution (10 -4 ，10 -5 ，10 -6 ，10 -7 ) 100. Mu.L of each dilution was plated on LB solid medium plates, incubated at 37℃for 24h, three replicates for each dilution, and single colony purification was selected.
2. Screening of anti-Aspergillus flavus strains
2.1 cultivation of Aspergillus flavus and preparation of spore liquid: inoculating Aspergillus flavus stored in a laboratory onto a PDA inclined plane, and culturing for 6d at 28 ℃; after the spores are fully formed, adding 4mL of sterile physiological saline to gently scrape the aspergillus flavus spores on the inclined plane to obtain spore suspension, transferring the spore suspension into a new sterile tube, counting by a blood cell counting plate after full oscillation, and preparing the concentration of 10 7 And (3) keeping the mixture at 4 ℃ for later use; the Aspergillus flavus is a commercial product, and the specific strain number is Aspergillusflavus ATCC28539; the aspergillus flavus colony is yellow green, loose, white and transparent in mycelium, has partitions and branches, and is loose and radial;
2.2 preliminary screening of anti-Aspergillus flavus strains: uniformly coating 100 mu L of the mould spore liquid obtained in the step 2.1 on a PDA flat plate; then the colony obtained by separation and purification in the step 1 is planted on a flat plate, the number of the colony seeds on each flat plate is moderate, and each colony is parallel; after culturing at 28 ℃ for 48 hours, observing whether a bacteriostasis zone exists around a test bacterial colony, and screening to obtain a primary screening strain, wherein the primary screening strain has good bacteriostasis effect, and the two strains are respectively numbered as B-301 and B-332;
2.3 re-screening of anti-Aspergillus flavus strains: taking a loop of bacterial strain with antibacterial activity obtained by the primary screening of the step 2.2 by using an inoculating loop, inoculating the bacterial strain into an LB liquid culture medium, performing shaking culture at 37 ℃ and 180r/min for 24 hours, centrifuging the culture solution for 10 minutes by 8000r/min, and collecting supernatant for later use;
uniformly coating 100 mu L of the mould spore liquid prepared in the step 2.1 on a PDA flat plate, placing three oxford cups on each plate, and respectively preparing test groups added with 100 mu L and 200 mu L of the supernatant obtained in the step 2.3 and a control group added with 100 mu L of physiological saline; finally, placing all the plates in a 28 ℃ incubator for culturing for 48-72 hours, observing whether a bacteriostasis zone exists around the test strain, and measuring the diameter of the bacteriostasis zone to obtain a re-screening result, wherein the specific re-screening result is shown in table 1 and fig. 1, and the strain with the number of B-332 can be seen to have a larger bacteriostasis zone.
TABLE 1
3. Screening of antibacterial lipopeptide high-producing strains
Taking a loop of the strain (number is B-332) with the largest antibacterial circle screened in the step 2.3 by using an inoculating loop, inoculating the strain into an LB liquid culture medium, performing shake culture for 18h at 37 ℃ and 180r/min, inoculating the strain into a fermentation culture medium according to 2% of inoculating quantity (volume fraction), and performing fermentation culture for 48h at 30 ℃ and 180 r/min; centrifuging the fermented fermentation liquor, collecting supernatant, adding 10 mu L of hydrochloric acid solution with the concentration of 6mol/L into 1mL of supernatant, regulating the pH to 2-2.5, stirring and mixing uniformly, finding that sediment is generated, and standing at the temperature of 4 ℃ for 12 hours; the precipitate was collected by centrifugation and dissolved in methanol, then filtered with a 0.2 μm filter membrane, and the filtered filtrate was analyzed for its components using a liquid chromatography-mass spectrometry analyzer (high performance liquid chromatography/2695 control panel 2998PDA, waters company, usa), wherein the conditions of liquid chromatography were: sample injection volume: 15. Mu.L; column temperature: 30 ℃; DAD detector detection range: 190-640nm; detection wavelength: 200nm; mobile phase: ultrapure water and acetonitrile, wherein item a is ultrapure water and item B is acetonitrile; the gradient elution conditions were: 0-1min,5% B-5% B;1-20min, 5-95% B; maintaining 95% B for 20-25 min; the mass spectrum conditions are as follows: ion source: electrospray ionization (ESI); atomization gas temperature: 350 ℃; spray pressure: 35psig; positive and negative ion capillary voltage: 3500V; dry gas flow rate: 8L/min; ion scan range (m/z): 100-2000;
the components in the filtrate were subjected to structural identification and were compared in literature (1, li Baoqing, deer Xiuyun, guo Qinggang, etc.. Separation and identification of lipopeptides produced by Bacillus subtilis BAB-1 and volatile matters [ J ]. Chinese agricultural science, 2010,43 (17): 3547-3554..2, jasim B, sreelakshmi K S, mathew J, et al, surfactin, iturin, and fengycinbiosynthesis by endophytic Bacillus sp.from Bacopa monnieri [ J ]. Microbial Ecology,2016,72 (1): 106-119.), and the obtained compound was confirmed to be bacitracin D (Bacillomycin D), and the peak areas of the liquid phases were compared to screen out the strain of the highly produced antimicrobial lipopeptide (bacitracin D). The strain bacterial liquid after re-screening is stored at the temperature of minus 80 ℃.
4. Final screening results
Through the screening process, a strain with obvious inhibition effect on aspergillus flavus is finally obtained, and the strain has the performance of high-yield antibacterial lipopeptide (bacitracin D). For ease of recording and management, the inventors named it: strain BYA-KC-4.
The antibacterial chart of the action 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 aspergillus flavus is shown in fig. 2, the reference numeral 1 in fig. 2 is an ethanol control with the volume fraction of 95%, the reference numeral 2 is an ethanol solution of the active antibacterial lipopeptide (wherein the solvent is ethanol with the volume fraction of 95%), and the prepared antibacterial lipopeptide has remarkable inhibition effect on the aspergillus flavus can be seen from fig. 2.
FIG. 3 is a liquid chromatogram of the analysis of the secondary metabolite produced by strain BYA-KC-4 in step 3 using a LC-MS analyzer, from which three strong absorption peaks a, b, C within 16-20min are mainly C of bacitracin D (Bacillomycin D) differing by one subunit 14 、C 15 、C 16 Homologs having molecular weights 1031.55, 1045.56 and 1059.57, respectively; as can also be seen from FIG. 3, other peaks include antibacterial lipopeptide components such as lysin (Bacilysin), fencine (Fengycin), and surfactant (Surfactan).
Furthermore, the inventor also carries out the test of the genetic stability of the antibacterial lipopeptide produced by the strain BYA-KC-4, the test result is shown in figure 4, and the inventor continuously subcultures the strain for 9 times, so that the performance of the strain is still stable, and the antibacterial activity and the production capacity of the antibacterial lipopeptide (bacitracin D) are still unchanged.
EXAMPLE 2 morphological characterization of Strain BYA-KC-4 and molecular biological characterization
Morphology feature (one):
diluting the strain BYA-KC-4 bacterial liquid obtained by re-screening by 100-1000 times, coating the bacterial liquid on an LB plate, and culturing for 24 hours at 37 ℃ to obtain bacterial colonies, as shown in figure 5; the observation shows that the bacterial colony of the strain BYA-KC-4 is round, smooth in surface, raised, opaque, milky white, neat in edge, has a certain viscosity and has a diameter of l-2 cm; gram positive staining; spores are 0.6-0.9 (width). Times.1.0-1.5 μm (width), oval or oval, and the bacterial cells do not obviously expand after the spores are formed.
(II) physiological and biochemical characteristics:
the strain has positive contact enzyme reaction and VP reaction, and can reduce nitrate and hydrolyze starch and gelatin. Can grow normally in liquid culture medium containing 1% -7% NaCl, and the specific physiological and biochemical characteristics are shown in the table 2 below.
TABLE 2 results of physiological and biochemical index measurements
Note that: ++, positive result: -, indicates that the result is negative.
(III) molecular biology identification:
PCR amplification is carried out on the cultured strain BYA-KC-4 bacterial liquid to obtain a 16S rDNA gene sequence, blast comparison is carried out on NCBI, the sequence is searched in a GenBank database, sequence homology analysis is carried out, sequencing results are seen in sequence SEQ ID NO.1, and the strain BYA-KC-4 is determined to be bacillus subtilis (Bacillus subtilis).
The bacillus subtilis BYA-KC-4 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation address of: the collection date is 2022, 9 and 19 days, and the collection number is CGMCC No.25758.
The 16S rDNA sequencing result of the strain is shown as SEQ ID NO.1, and specifically comprises the following steps:
example 3 method for achieving high yield of antibacterial lipopeptid 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 minus 80 ℃, freezing and thawing the glycerol freezing tube in an ice bath, inoculating the thawed bacterial liquid into an LB solid slant culture medium by an inoculating loop, and culturing for 24 hours at the temperature of 30 ℃ to activate strains;
1.2 seed culture: inoculating a ring of inclined plane lawn to 10mL of LB liquid culture medium (using a triangular shake flask with the volume of 50 mL) by using an inoculating loop after inclined plane culture, and culturing for 16-18 h at 30 ℃ and 180r/min by using a shaking table to obtain first-stage seed liquid;
1.3 seed expansion: inoculating 1mL of the primary seed solution to 50mL of LB liquid medium (using a triangular shake flask with the volume of 250 mL), namely, 2% of inoculating amount (volume fraction), and culturing for 16-18 h at 30 ℃ in a shaking table of 180r/min to obtain the expanded seed solution, wherein the concentration of the bacillus subtilis BYA-KC-4 is 1x10 9 cfu/mL；
1.4 fermentation culture: 10mL of the expanded seed solution was inoculated into 200mL of a fermentation medium (using a flask with a volume of 1L), i.e., the inoculum size (volume fraction) was 5%, and cultured at 30℃for 48 hours in a shaker 180r/min to obtain 200mL of a fermentation broth having a bacterial concentration of about 10% (volume fraction).
2. Method for producing active antibacterial lipopeptid by using bacillus subtilis BYA-KC-4
2.1, taking the bacillus subtilis fermentation liquor, centrifuging for 10min at a rotating speed of 8000r/min, removing bacterial precipitate, and combining and collecting fermentation supernatant;
2.2 taking 1L of fermentation supernatant, regulating the pH of the supernatant to 2.5 by using hydrochloric acid solution with the concentration of 6mol/L, wherein precipitation is generated at the moment, and placing the supernatant in a refrigerator at the temperature of 4 ℃ overnight to ensure that the precipitation is complete; gently pouring out part of the supernatant liquid in the next day, centrifuging the rest suspension liquid for 10min at the rotating speed of 8000r/min, and collecting sediment, wherein about 8-10 g sediment is contained in 1L fermentation supernatant liquid;
2.3 repeatedly dissolving and extracting the obtained precipitate with 50mL of absolute ethyl alcohol for 3 times, and combining the extracting solutions; then, a microporous filter membrane (a mixed membrane, a mixed fiber microporous filter membrane water system/nylon filter membrane organic system, 50mm in specification, 0.22 mu m in thickness and 0.8 mu m in thickness, which is purchased from Shanghai Xinya purification device factory) with the size of 0.22 mu m is selected for filtering and impurity removing the ethanol extract to obtain a clear extract (about 120-130 mL);
2.4, decompressing and distilling the ethanol solvent of the extracting solution by a rotary evaporator at 55 ℃ until the ethanol solvent is completely evaporated; scraping and grinding solid matters on the wall of the rotary steaming bottle into powder to obtain an antibacterial lipopeptide extract, calculating the yield of the extract in each 1L of fermentation supernatant to be 550-780 mg, and calculating the content of the identified antibacterial lipopeptide in the extract by normalizing the area of a liquid chromatographic peak;
furthermore, the extract is subjected to separation and purification of target components by adopting a macroporous resin and silica gel column chromatography method, and the liquid phase and mass spectrum identification shows that the component of the bacitracin D in the identified antibacterial lipopeptide reaches more than 93 percent, specifically 93.36 percent, so that the method of the embodiment can realize the preparation of the high-yield antibacterial lipopeptide (bacitracin D).
EXAMPLE 4 degradation of zearalenone toxin Using the active antibacterial lipopeptides obtained with Bacillus subtilis BYA-KC-4
1. Sample treatment:
0.7g of the active antibacterial lipopeptide prepared in example 3 is dissolved in 6.3mL of PBS (0.01 mol/L) buffer solution, and centrifuged at 10000r/min for 10min, and the supernatant is collected as a sample solution; taking 1200 mu L of sample solution, adding 300 mu L (2500 ppb) of zearalenone toxin into the sample solution to perform a reaction as a test group, and adding 300 mu L (2500 ppb) of zearalenone toxin into 1200 mu L of PBS buffer solution as a control group; the pH of the reaction system was adjusted to 6.2, and the test group and the control group were reacted at 37℃for 2 hours, 5 hours, 12 hours, 24 hours, 48 hours and 72 hours, respectively, with 200. Mu.L of each sample.
2. Corn gibberellin toxin content detection (ELISA method, the following kit is the ELISA corn gibberellin toxin kit of Beijing Bunge, wherein the dosage and detection method of each reagent refer to the specific use method in the kit):
2.1, taking the kit out of the refrigerator at the temperature of 4 ℃ and placing the kit at the room temperature for 30min; the microwells corresponding to the samples and standards were numbered on a 96-well plate, with 2 wells per well in parallel.
2.2 sucking 20 mu L of standard substance or sample, adding into corresponding microwells, adding 100 mu L of enzyme conjugate working solution, gently shaking, mixing, covering with cover film, and reacting in a light-shielding environment at 25deg.C for 15min.
2.3 carefully uncovering the cover plate film and spin-drying the liquid in the holes; adding 250 mu L of deionized water into each hole, fully washing for 4-5 times, pouring out the washing liquid in the hole of the hole at intervals of 10s each time, and beating with absorbent paper; if bubbles exist after the drying, the sterilized gun head is used for puncturing.
2.4 adding 50 mu L of substrate A solution and 50 mu L of substrate B solution into each hole, gently shaking and mixing, and carrying out light-shielding reaction at 25 ℃ for 5min; after the reaction, blue with different shades can be seen, and the darker the color is, the smaller the concentration of the representative toxin is, for example, the lighter the overall color is, and the reaction time can be prolonged to 7min.
2.5 adding 50 mu L of stop solution to each well, gently shaking and mixing to terminate the reaction.
2.6 absorbance values were measured per well with a microplate reader at 450 nm.
3. And (3) manufacturing a standard curve:
preparing a zearalenone toxin standard solution, wherein the concentration of the zearalenone toxin standard solution is respectively 0ppb, 20ppb, 80ppb, 240ppb and 1000ppb; the average value of absorbance values of each concentration standard solution and sample of the obtained zearalenone toxin (E Red with red color ) Divided by the absorbance value (E) of the first standard (standard 0) 0 red ) Multiplying by 100%, namely the percentage absorbance value;
percent absorbance value (%) =e Red with red color /E 0 red ×100％；
Drawing a standard curve by taking the percentage absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (ppb) of the zearalenone toxin standard substance as an abscissa to obtain a regression equation; and (5) taking the percent absorbance of the sample into an equation to obtain the concentration of zearalenone toxin in the sample.
4. The degradation rate calculation formula:
zearalenone toxin degradation rate (%) = (a) 0 -A)/A 0 *100；
A 0 (ppb) mean zearalenone toxin concentration of control group;
a (ppb) corresponds to the average value of the zearalenone toxin concentration per hour;
5. detection result:
as shown in FIG. 6, it can be seen that when zearalenone toxin is degraded by using the active antibacterial lipopeptid produced by bacillus subtilis BYA-KC-4, the degradation effect is increased with time; the degradation rate of the active antibacterial lipopeptide of the bacillus subtilis BYA-KC-4 on zearalenone toxin is 27% after the reaction for 2 hours; reacting for 5 hours, wherein the degradation rate of the zearalenone toxin is 58%; reacting for 12 hours, wherein the degradation rate of the zearalenone toxin is 77%; reacting for 24 hours, wherein the degradation rate of the zearalenone toxin is 84%; reacting for 48 hours, wherein the degradation rate of the zearalenone toxin is 90%; the degradation rate of zearalenone toxin is 92% after the reaction is carried out for 72 hours.
EXAMPLE 5 degradation of vomitoxin by active antibacterial lipopeptides obtained using the Bacillus subtilis BYA-KC-4
1. Sample treatment:
0.7g of the active antibacterial lipopeptide prepared in example 3 is dissolved in 6.3mL of PBS (0.01 mol/L) buffer solution, and centrifuged at 10000r/min for 10min, and the supernatant is collected as a sample solution; taking 1200 mu L of sample solution, adding 300 mu L (10000 ppb) of vomitoxin as a test group for reaction, and adding 300 mu L (10000 ppb) of vomitoxin into 1200 mu L of PBS buffer solution as a control group; the pH of the reaction system was adjusted to 6.2, and the test group and the control group were reacted at 37℃for 2 hours, 5 hours, 12 hours, 24 hours, 48 hours and 72 hours, respectively, with 200. Mu.L of each sample.
2. Vomitoxin content detection (ELISA method, the following kit is the ELISA vomitoxin kit of Beijing Bualobang, wherein the dosage and detection method of each reagent refer to the specific use method in the kit):
2.1, taking the kit out of the refrigerator at the temperature of 4 ℃ and placing the kit at the room temperature for 30min; the microwells corresponding to the samples and standards were numbered on a 96-well plate, with 2 wells per well in parallel.
2.2 sucking 20 mu L of standard substance or sample, adding into corresponding microwells, adding 100 mu L of enzyme conjugate working solution, gently shaking, mixing, covering with cover film, and reacting in a light-shielding environment at 25deg.C for 15min.
2.3 carefully uncovering the cover plate film and spin-drying the liquid in the holes; adding 250 mu L of deionized water into each hole, fully washing for 4-5 times, pouring out the washing liquid in the hole of the hole at intervals of 10s each time, and beating with absorbent paper; if bubbles exist after the drying, the sterilized gun head is used for puncturing.
2.4 adding 50 mu L of substrate A solution and 50 mu L of substrate B solution into each hole, gently shaking and mixing, and carrying out light-shielding reaction at 25 ℃ for 5min; after the reaction, blue with different shades can be seen, and the darker the color is, the smaller the concentration of the representative toxin is, for example, the lighter the overall color is, and the reaction time can be prolonged to 7min.
2.5 adding 50 mu L of stop solution to each well, gently shaking and mixing to terminate the reaction.
2.6 absorbance values were measured per well with a microplate reader at 450 nm.
3. And (3) manufacturing a standard curve:
the vomitoxin standard solution was prepared at concentrations of 0ppb, 150ppb, 500ppb, 1500ppb, and 5000ppb, respectively. By a means ofAverage value of absorbance values of each concentration standard solution and sample of vomitoxin obtained (E Vomiting type ) Divided by the absorbance value (E) of the first standard (standard 0) 0 vomiting ) Multiplying by 100%, namely the percentage absorbance value;
percent absorbance value (%) =e Vomiting type /E 0 vomiting ×100％；
Drawing a standard curve by taking the percentage absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (ppb) of the vomitoxin standard substance as an abscissa to obtain a regression equation; and carrying the percent absorbance of the sample into an equation to obtain the vomitoxin concentration in the sample.
4. The degradation rate calculation formula:
vomitoxin degradation rate (%) = (B 0 -B)/B 0 *100；
B 0 (ppb) mean vomitoxin concentration in control group;
b (ppb) is the average value of vomitoxin concentration per hour;
5. detection result:
as shown in FIG. 7, it can be seen that when the active antibacterial lipopeptides produced by the Bacillus subtilis BYA-KC-4 of the present invention are used for degrading vomitoxin, the degradation increases with time; the reaction is carried out for 2 hours, and the degradation rate of the active antibacterial lipopeptide of the bacillus subtilis BYA-KC-4 to vomit toxin is 23%; reacting for 5 hours, wherein the degradation rate of vomitoxin is 34%; reacting for 12 hours, wherein the degradation rate of vomitoxin is 55%; reacting for 24 hours, wherein the degradation rate of vomitoxin is 61%; reacting for 48 hours, wherein the degradation rate of vomitoxin is 67%; the reaction is carried out for 72 hours, and the degradation rate of vomitoxin is 81%.
EXAMPLE 6 degradation of aflatoxin B Using the active antibacterial lipopeptides obtained by Bacillus subtilis BYA-KC-4 1
1. Sample treatment:
0.7g of the active antibacterial lipopeptide prepared in example 3 is dissolved in 6.3mL of PBS (0.01 mol/L) buffer solution, and centrifuged at 10000r/min for 10min, and the supernatant is collected as a sample solution; a1200. Mu.L sample solution was taken and 300. Mu.L (200 ppb) of aflatoxin B was added thereto 1 Performing a reaction as a test group; control group 1200. Mu.L PBS buffer was added300 mu L (200 ppb concentration) of aflatoxin B 1 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the reaction system was adjusted to 6.2, and the test group and the control group were reacted at 37℃for 2 hours, 5 hours, 12 hours, 24 hours, 48 hours and 72 hours, respectively, with 200. Mu.L of each sample.
2. Aflatoxin B 1 Content detection (ELISA method, ELISA aflatoxin B prepared from Shenzhen Yirui biotechnology using the following kit 1 Kit, wherein the amount of each reagent and the detection method are as follows in the specific use method of the kit):
2.1, taking the kit out of the refrigerator at the temperature of 4 ℃ and placing the kit at the room temperature for 30min; the microwells corresponding to the samples and standards were numbered on a 96-well plate, with 2 wells per well in parallel.
2.2 sucking 20 mu L of standard substance or sample, adding into corresponding microwells, adding 100 mu L of enzyme conjugate working solution, gently shaking, mixing, covering with cover film, and reacting in a light-shielding environment at 25deg.C for 15min.
2.3 carefully uncovering the cover plate film and spin-drying the liquid in the holes; adding 250 mu L of deionized water into each hole, fully washing for 4-5 times, pouring out the washing liquid in the hole of the hole at intervals of 10s each time, and beating with absorbent paper; if bubbles exist after the drying, the sterilized gun head is used for puncturing.
2.4 adding 50 mu L of substrate A solution and 50 mu L of substrate B solution into each hole, gently shaking and mixing, and carrying out light-shielding reaction at 25 ℃ for 5min; after the reaction, blue with different shades can be seen, and the darker the color is, the smaller the concentration of the representative toxin is, for example, the lighter the overall color is, and the reaction time can be prolonged to 7min.
2.5 adding 50 mu L of stop solution to each well, gently shaking and mixing to terminate the reaction.
2.6 absorbance values were measured per well with a microplate reader at 450 nm.
3. And (3) manufacturing a standard curve:
preparation of aflatoxin B 1 The standard solutions had concentrations of 0ppb, 2ppb, 5ppb, 20ppb, and 50ppb, respectively. The aflatoxin B obtained 1 Average value of absorbance value of each concentration standard solution and sample (E Yellow colour ) Divided by the absorbance value (E) of the first standard (standard 0) 0 yellow ) Multiplying by 100%, namely the percentage absorbance value;
percent absorbance value (%) =e Yellow colour /E 0 yellow ×100％；
The standard percent absorbance is taken as the ordinate, and aflatoxin B is taken as the ordinate 1 The logarithm of the standard concentration (ppb) is the abscissa, and a standard curve is drawn to obtain a regression equation; bringing the percent absorbance of the sample into an equation to obtain aflatoxin B in the sample 1 Concentration.
4. The degradation rate calculation formula:
aflatoxin B 1 Degradation rate (%) = (C 0 -C)/C0*100；
C 0 Control group aflatoxin B 1 Concentration average;
c (ppb) corresponds to aflatoxin B per hour 1 Concentration average;
5. detection result:
as shown in FIG. 8, it can be seen that the active antibacterial lipopeptides produced by using the bacillus subtilis BYA-KC-4 of the present invention are effective against aflatoxin B 1 When degradation is carried out, the degradation effect is increased along with the change of time; reaction for 2h, active antibacterial lipopeptide of bacillus subtilis BYA-KC-4 against aflatoxin B 1 The degradation rate of (2) is 25%; reacting for 5h, for aflatoxin B 1 The degradation rate of (2) is 31%; reacting for 12h, for aflatoxin B 1 The degradation rate of (2) is 45%; reacting for 24h, for aflatoxin B 1 The degradation rate of (2) is 60%; reacting for 48h, for aflatoxin B 1 The degradation rate of (2) is 79%; reacting for 72h, against aflatoxin B 1 The degradation rate of (2) was 84%.
In conclusion, the invention proves that the screened bacillus subtilis BYA-KC-4 has the capability of efficiently degrading various mycotoxins and the performance of high-yield active antibacterial lipopeptid, and based on the bacillus subtilis, the bacillus subtilis has good application value in the aspects of developing novel biological detoxication agents, feed additives and the like, reducing pollution of feed or processing raw materials to mycotoxins and the like.
While the invention has been illustrated and described with specific examples, it will be appreciated that the embodiments of the invention are not limited by the examples, but are intended to cover various modifications, adaptations, substitutions, combinations, and simplifications without departing from the spirit and principles of the invention.
Claims (7)
1. High yield bacillus subtilis strain of antibacterial lipopeptideBacillus subtilis) Strain BYA-KC-4, characterized in that the bacillus subtilis has a deposit number of: CGMCC No. 25758;
the main component of the antibacterial lipopeptide is bacillus toxin D.
2. Use of the antibacterial lipopeptide high-yield bacillus subtilis strain BYA-KC-4 according to claim 1 for preparing antibacterial lipopeptid, characterized by being used for preparing antibacterial lipopeptid by a biological fermentation method.
3. A method for producing an antibacterial lipopeptide by fermenting the antibacterial lipopeptide-producing bacillus subtilis strain BYA-KC-4 according to claim 1, comprising the following steps:
(1) Activating strains: inoculating bacillus subtilis into an LB solid culture medium, and culturing for 12-24 hours at 28-32 ℃ to obtain an activated strain;
(2) Seed culture: inoculating the activated strain to an LB liquid culture medium, and culturing for 16-18 hours at 28-32 ℃ to obtain primary seed liquid;
(3) And (3) performing expansion 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 seed liquid;
(4) Fermentation culture: inoculating the seed expanding culture liquid to a fermentation medium, and culturing for 12-48 hours at 28-32 ℃ to obtain a fermentation liquid;
(5) Centrifuging the fermentation liquor obtained in the step (4) to obtain fermentation supernatant, regulating 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 the supernatant to obtain suspension, and centrifuging to obtain precipitate;
(6) Extracting the precipitate obtained in the step (5) with absolute ethyl alcohol to obtain an extracting solution, filtering to remove impurities, and distilling the ethanol solvent under reduced pressure at 50-60 ℃ to obtain a solid product, namely the antibacterial lipopeptide.
4. The method according to claim 3, wherein the seed liquid for propagation obtained in the step (3) has a concentration of 1X10 9 cfu/mL~2x10 9 cfu/mL。
5. A method according to claim 3, wherein the inoculum size of the fermentation culture in step (4) is 2-5%.
6. The use of the antibacterial lipopeptide high-yield bacillus subtilis strain BYA-KC-4 in preparing a mycotoxin degrading medicament, which is characterized in that the mycotoxin is zearalenone toxin, vomit toxin and aflatoxin B 1 One or more of the following.
7. The use of the antibacterial lipopeptide high-yield bacillus subtilis strain BYA-KC-4 in preparing a feed additive for degrading mycotoxins, which is characterized in that the mycotoxins are zearalenone toxin, vomitoxin and aflatoxin B 1 One or more of the following.
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| 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 |
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| 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 |
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