Biological agent for efficiently degrading mycotoxin, method and application

Abstract

The invention discloses a biological agent for efficiently degrading mycotoxin, a method and application. Belongs to the technical field of enzyme biological preparations. The novel laccase is obtained from a Ganoderma lucidum culture solution and is named as GL-Lac, the amino acid sequence of the novel laccase is shown as SEQ ID NO.1, the novel laccase can effectively degrade AFB1, ZEN, DON, T-2 and other mycotoxins, non-phenolic toxin-AFB1 can be efficiently oxidized without additional mediators, in addition, by means of a composite induction strategy, the degradation rates of AFB1, ZEN, DON and T-2 toxins are remarkably increased and reach 80% or above, 90% or above, 78% or above and 75% or above respectively, and the novel laccase is suitable for industrial production. And a technical support is provided for degradation of mycotoxin.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme biological agents, and more particularly to a biological agent, method and application for efficiently degrading mycotoxins. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced by molds during growth. Currently, various mycotoxins have been discovered, which seriously endanger the health of animals and humans. The mycotoxins that widely exist in feed and have greater harm mainly include: aflatoxin, zearalenone (ZEN), vomitoxin, T-2 toxin, ochratoxin, fumonisin, alternaria toxin and their derivatives, etc. Therefore, effectively controlling mycotoxins in feed is of great significance to the safety of livestock, poultry and human lives.

[0003] Existing degradation technologies for mycotoxins mainly include physical adsorption, chemical treatment and biological detoxification methods. The biological detoxification of microorganisms or enzymes has received extensive attention due to its safety and high efficiency. Most existing mycotoxin-degrading enzymes have defects such as low catalytic efficiency, low expression level, poor temperature and pH tolerance, which limit their industrial application. Therefore, it is necessary to explore new enzymes with high catalytic activity and high temperature and pH tolerance. Laccase, as one of them, has attracted much attention due to its broad-spectrum oxidation ability, but the existing laccase has the following problems: low degradation efficiency, especially poor effect on non-phenolic toxins (such as AFB1); insufficient enzyme production and high industrial production cost; lack of specific laccase for multiple toxins.

[0004] Therefore, how to further develop a new laccase with good degradation effect on non-phenolic mycotoxins and a wide range of degradation targets is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a biological agent, method and application for efficiently degrading mycotoxins.

[0006] The first object of the present application is to provide: a biological agent for efficiently degrading mycotoxins, including laccase GL-Lac; the amino acid sequence of the laccase GL-Lac is shown in SEQ ID NO.1.

[0007] As a preferred technical solution, the mycotoxins include at least one of aflatoxin B1, zearalenone, vomitoxin, and T-2 toxin; the laccase GL-Lac can degrade at least one of aflatoxin B1, zearalenone, vomitoxin, and T-2 toxin without relying on any mediator.

[0008] As a preferred technical solution, the laccase GL-Lac is derived from Ganoderma lucidum ACCC 53264.

[0009] As a preferred technical solution, the laccase GL-Lac is prepared by the following method: Ganoderma lucidum ACCC53264 is cultured in an induction medium until the mid-logarithmic growth phase, an inducer composition is added, and induction culture is carried out. Then, centrifugation, separation, and precipitation are performed to obtain the laccase GL-Lac.

[0010] As a preferred technical solution, the components and ratios of the induction medium are as follows: corn flour 20 g / L, peptone 2 g / L, KH2PO4 1 g / L, and MgSO4·7H2O 0.5 g / L; pH 5.0.

[0011] As a preferred technical solution, the following weights of the inducer composition are added to each 1 L of the medium: 0.3 mM coumarin, 0.5 mM acetosyringone, 2 mM Cu 2+ 、0.05 mM β-estradiol, 2.0 g / L lignosulfonate, 0.10 mM Mn 2+ 、10 μg / L 3-acetyl-DON, and 0.5 mM ferulic acid.

[0012] As a preferred technical solution, the biological agent for degrading mycotoxins further includes other active ingredients and / or other physiologically acceptable carriers.

[0013] Another object of the present application is to provide: the application of the above biological agent in degrading mycotoxins in feed.

[0014] Another object of the present application is to provide: a method for degrading mycotoxins in feed using laccase GL-Lac, the amino acid sequence of the laccase GL-Lac is as shown in SEQ ID NO.1; The mycotoxins include at least one of aflatoxin B1, zearalenone, vomitoxin, and T-2 toxin.

[0015] Through the above technical solutions, compared with the prior art, the present invention has the following beneficial effects: The present invention obtains a novel laccase (named GL-Lac) from the Ganoderma lucidum culture solution through a composite induction strategy, which has the amino acid sequence as shown in SEQ ID NO:1 and has the following characteristics: The degradation rates of AFB1, ZEN, DON, and T-2 toxins reach ≥80%, ≥90%, ≥78%, and ≥75% respectively; it can efficiently oxidize non-phenolic toxins, such as AFB1, without the addition of an external mediator. Detailed implementation manners

[0016] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0017] Example 1

[0018] Preparation and extraction of laccase GL-Lac

[0019] (1) Strain activation: Take Ganoderma lucidum ( Ganoderma lucidum ) ACCC 53264, inoculate it onto a PDA plate containing 0.5 mM CuSO4, and culture it at 28 °C for 5 days until the mycelium covers the plate; (2) Seed liquid culture: Punch out 5 mycelial cakes with a diameter of 5 mm (the active area of the mycelium at the edge) from the activated plate, inoculate them into a 250 mL conical flask (containing 100 mL of wheat bran extract medium), and culture them at 28 °C and 150 rpm for 3 days. When the diameter of the mycelial balls is 1-2 mm and the culture solution is slightly turbid (OD 600 is 1.2-1.5), stop the culture; Wheat bran extract medium: Wheat bran extract 15 g / L (filter the supernatant after boiling for 30 min) Ammonium tartrate 3 g / L KH2PO4 1 g / L MgSO4·7H2O 0.5 g / L CuSO4·5H2O 0.125 g / L (0.5 mM) pH 5.5 (adjusted with 0.1 M HCl or NaOH); The wheat bran extract provides a complex carbon source (hemicellulose, lignin fragments) to simulate the natural degradation environment; Low nitrogen (ammonium tartrate) promotes the accumulation of secondary metabolites (such as laccase).

[0020] (3) Enzyme production induction: Transfer the seed liquid to the induction medium (loading volume 50 mL / 250 mL conical flask) at an inoculation amount of 10%, culture it at 28 °C and 200 rpm. When it reaches the mid-logarithmic growth phase, add the inducer composition (to avoid initial growth inhibition) and continue the culture. During this period, take samples and measure the laccase activity by the ABTS method. When the laccase activity is ≥500 U / L on the 7th day of culture, stop the culture: Induction medium (pH 5.0, adjusted before sterilization): Corn flour 20 g / L (gelatinize at 80 °C for 30 min and then filter); Peptone 2 g / L; KH2PO4 1 g / L; MgSO4·7H2O 0.5 g / L; The following weight of inducer composition is added to each 1 L of the culture medium: 0.3 mM coumarin, 0.5 mM acetosyringone, 2 mM Cu 2+ , 0.05 mM β-estradiol, 2.0 g / L lignosulfonate, 0.10 mM Mn 2+ , 10 μg / L 3-acetyl-DON and 0.5 mM ferulic acid.

[0021] Laccase purification: 1) Preparation of crude enzyme solution: The culture solution was centrifuged at 8000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.45 μm filter membrane to obtain the crude enzyme solution; 2) Ammonium sulfate fractional precipitation: Take the crude enzyme solution prepared in step 1), slowly add solid (NH4)2SO4 to it until 30% saturation, and then stir at 4 °C for 1 h; centrifuge at 10000 rpm for 20 min, discard the precipitate (impure protein); take the supernatant and add (NH4)2SO4 to 70% saturation, and centrifuge to collect the precipitate; the precipitate was redissolved with 10 mM sodium citrate buffer (pH 5.0) containing 0.1 mM CuSO4 and dialyzed to remove salts; 3) Ion exchange chromatography (DEAE-Sepharose FF): Column equilibration: 20 mM Tris-HCl buffer (pH 7.5); Sample loading: The dialyzed sample was loaded onto the column at a flow rate of 1 mL / min; Elution: Linear gradient elution with 0 - 1 M NaCl, and collect the laccase activity peak (in the range of 0.3 - 0.5 M NaCl); Concentration: Ultrafiltration centrifugation (10 kDa molecular weight cut-off) to obtain laccase GL-Lac.

[0022] ) Purity verification: SDS-PAGE: Single band (65 kDa), purity ≥ 95% (gel scanning analysis); Specific activity: ≥ 50 U / mg (ABTS method, pH 3.0).

[0023] Analyze the amino acid sequence of laccase GL-Lac, and its amino acid sequence is as follows: SRHWHGFFQEGSSWADGPVGVTQCPIAPGDSFLYRFKVPDQAGTFWYHSMAKFQSLLSCVTLLFAASAHAGIGPKADLTISNANIAPDGYTRAAVVVNGVFPGPLITGNKGDRFQLNVIDQLTNHTMLKTTSIHWHGFFQKGTNWADGPAFINQCPIASGHSFLYDFQVPDQAGTFWYHSHLSTQYCDGLRGPFVVYDPKDPLKGLYDVDNDSTVITLSDWYHVAARLGPSFPLGSDSTLINGLGRSTTNATAGLAVINVTQGKRYRFRLVSLSCDPNYTFSIDGHDSSVIEADGIATQPVTANAIQIFSAQRYSFVLTKNQTIGNYWIRANPSFGNIGFTTGINSAILRYSGADPIEPTTAQQTTQNLLNEVDLHPFVAMQTPGRATQGGTDVAINMVFNFNGSNFFINNASFTPPTVPVLLQILSGAQAAQDLLPSGSVYTLPINKSSELTFPATVNAPGAPHPFHLHGHSFAVVRSAGSTEYNYNNPVWRDVVSTGTPAAGDNVTIRFQTDNPGPWFLHCHIDFHLEAGFAVVFAEDTADTSLANHVPQAWSDLCP TYDALSADDH, SEQ ID NO.1.

[0024] Example 2 Degradation of mycotoxins by laccase GL-Lac

[0025] GL-Lac (500 U / L) and toxin (1 μg / mL AFB1 / ZEN / DON / T-2) were reacted at pH 4.5 and 40 °C for 24 h, and the degradation rates of different toxins were measured. The specific experimental procedure is as follows: Preparation of laccase stock solution: Purified GL-Lac laccase (specific activity ≥ 50 U / mg, dissolved in 10 mM sodium citrate buffer at pH 5.0); Preparation of toxin stock solution: Aflatoxin B1 (AFB1, 1 μg / mL dissolved in methanol); Zearalenone (ZEN, 1 μg / mL dissolved in acetonitrile); Deoxynivalenol (DON, 1 μg / mL dissolved in water); T-2 toxin (1 μg / mL dissolved in methanol); Reaction buffer: 0.1 M citric acid-phosphate buffer (pH 4.5).

[0026] The detection system is set as shown in Table 1: Table 1 Detection system settings

[0027] Note: The mother liquor of the toxin is dried with nitrogen and redissolved in the buffer (to avoid inhibition of enzyme activity by organic solvents).

[0028] Control group: Without laccase (only buffer + toxin), used to calculate the degradation rate, with 3 replicates in each group; Add the above reaction system to a 1.5 mL centrifuge tube, vortex and mix well, place it in a constant temperature water bath shaker, and react with shaking at 40 °C and 150 rpm for 24 h; Add 100 μL of 1 M HCl to terminate the reaction (pH < 2.0), immediately place it in an ice bath, then centrifuge at 4 °C and 12,000 rpm for 5 min, and take the supernatant for detecting the degradation rate of the target toxin. The specific detection method is as follows: (1) Degradation rate of AFB1 (detected by HPLC-FLD) Chromatographic conditions: Chromatographic column: C18 column (4.6×250 mm, 5 μm); Mobile phase: water: methanol: acetonitrile (60:20:20, v / v / v); Flow rate: 1 mL / min; Detector: fluorescence (Ex 360 nm, Em 440 nm); Calculation: Degradation rate (%) = (1 - sample peak area / control peak area) × 100.

[0029] (2) Degradation rate of ZEN (detected by ELISA) Steps: Operate according to the instructions of the RIDASCREEN® ZEN kit to determine the residual ZEN concentration in the reaction solution; Standard curve range: 0.1 - 5 μg / L; Calculation: Degradation rate (%) = (1 - sample OD value / control OD value) × 100.

[0030] (3) Degradation rate of DON / T-2 toxin (detected by HPLC-UV) Chromatographic conditions: Detection wavelength: DON (220 nm), T-2 (210 nm); Mobile phase: acetonitrile: water (15:85, v / v); Calculation: The same as AFB1.

[0031] The detection results are shown in Table 2.

[0032] Table 2 Degradation rate and residue rate of different toxins

[0033] Result analysis: As can be seen from the content of Table 2, the degradation rates of the laccase prepared in this application for AFB1, ZEN, DON, and T-2 toxins reach ≥80%, ≥90%, ≥78%, and ≥75% respectively; the non-phenolic toxin - AFB1 can be efficiently oxidized without adding an external mediator.

[0034] Control Group 1

[0035] It is the same as the preparation method of the laccase in Example 1, except that during the cultivation process, the induction composition is not added.

[0036] Further determination was made on the degradation effect and residue rate of the product of Control Group 1 on different toxins. The determination method was the same as that in Example 2, and the determination results are shown in Table 3.

[0037] Table 3 Degradation rate and residue rate of different toxins

[0038] Result analysis: As can be seen from the content of Table 3, even without adding the induction composition, the degradation rates of the product for AFB1, ZEN, DON, and T-2 toxins reach ≥70%, ≥80%, ≥60%, and ≥60% respectively; the non-phenolic toxin - AFB1 can be efficiently oxidized without adding an external mediator.

[0039] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same and similar parts among the embodiments, reference can be made to each other.

[0040] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather should be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A biological agent for efficiently degrading mycotoxins, characterized in that, It includes laccase GL-Lac; the amino acid sequence of the laccase GL-Lac is as shown in SEQ ID NO.

1.

2. The biological agent for efficiently degrading mycotoxins according to claim 1, characterized in that, The mycotoxins include at least one of aflatoxin B1, zearalenone, deoxynivalenol, and T-2 toxin; the laccase GL-Lac can degrade at least one of aflatoxin B1, zearalenone, deoxynivalenol, and T-2 toxin without relying on any mediator.

3. The biological agent for efficiently degrading mycotoxins according to claim 1, characterized in that, The laccase GL-Lac is derived from Ganoderma lucidum ACCC 53264.

4. The biological agent for efficiently degrading mycotoxins according to claim 3, characterized in that, The laccase GL-Lac is prepared by the following method: Ganoderma lucidum ACCC 53264 is cultured in an induction medium until the mid-logarithmic growth phase, an inducer composition is added, and induction culture is carried out. Then, it is centrifuged, separated, and precipitated to obtain the laccase GL-Lac.

5. The biological agent for efficiently degrading mycotoxins according to claim 4, characterized in that, The components and ratios of the induction medium are as follows: corn flour 20 g / L, peptone 2 g / L, KH2PO4 1 g / L, and MgSO4·7H2O 0.5 g / L; pH 5.

0.

6. The biological agent for efficiently degrading mycotoxins according to claim 5, wherein Add an inducer composition with the following weights to every 1 L of the culture medium: 0.3 mM coumarin, 0.5 mM acetosyringone, 2 mM Cu 2+ , 0.05 mM β-estradiol, 2.0 g / L lignosulfonate, 0.10 mM Mn 2+ , 10 μg / L 3-acetyl-DON, and 0.5 mM ferulic acid.

7. The biological agent for efficiently degrading mycotoxins according to claim 1, characterized in that, It also includes other active ingredients and / or other physiologically acceptable carriers.

8. Use of the biological agent according to any one of claims 1-7 in degrading mycotoxins in feed.

9. A method for degrading mycotoxins in feed by using laccase GL-Lac, characterized in that, The amino acid sequence of the laccase GL-Lac is as shown in SEQ ID NO.1; The mycotoxins include at least one of aflatoxin B1, zearalenone, deoxynivalenol, and T-2 toxin.

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