A biological preparation, method and application for efficiently degrading mycotoxins

Abstract

This application discloses a biological preparation, method and application for efficiently degrading mycotoxins. It belongs to the technical field of enzyme biological preparations. Ganoderma lucidum A new type of laccase was obtained from the culture medium, named GL-Lac, whose amino acid sequence is shown in SEQ ID NO.1. It can effectively degrade mycotoxins such as AFB1, ZEN, DON and T-2, and can efficiently oxidize non-phenolic toxin ‑AFB1 without the addition of external mediators. In addition, through a composite induction strategy, the degradation rates of AFB1, ZEN, DON and T-2 toxins were significantly improved, reaching ≥80%, ≥90%, ≥78% and ≥75%, respectively, providing technical support for the degradation of mycotoxins.

Description

Technical Field

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

[0002] Mycotoxins are toxic secondary metabolites produced by mold during its growth. A wide variety of mycotoxins have been discovered, posing serious threats to animal and human health. Mycotoxins that are widely present in feed and pose significant risks include aflatoxins, zearalenone (ZEN), vomitoxin, T-2 toxin, ochratoxin, fumonisin, and alternaria toxins and their derivatives. Therefore, effective control of mycotoxins in feed is crucial for the safety of livestock, poultry, and humans.

[0003] Existing mycotoxin degradation technologies primarily include physical adsorption, chemical treatment, and biological detoxification. Microbial or enzymatic biological detoxification has attracted widespread attention due to its safety and efficiency. However, most existing mycotoxin-degrading enzymes suffer from low catalytic efficiency, low expression levels, and poor temperature and pH tolerance, limiting their industrial application. Therefore, it is necessary to identify new enzymes with high catalytic activity and high temperature and pH tolerance. Laccase, one of these enzymes, has attracted considerable attention due to its broad-spectrum oxidative capacity. However, existing laccases suffer from the following challenges: low degradation efficiency, particularly for non-phenolic toxins (such as AFB1); insufficient enzyme yield, resulting in high industrial production costs; and a lack of specific laccases for multiple toxins.

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

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

[0006] The first object of the present application is to provide: a biological preparation for efficiently degrading mycotoxins, comprising 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: culturing Ganoderma lucidum ACCC53264 to mid-logarithmic growth stage in an induction medium, adding an inducer composition, inducing the culture, and centrifuging, separating, and precipitating to prepare laccase GL-Lac.

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

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

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

[0013] Another object of the present application is to provide: application of the above-mentioned biological preparation 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, wherein the amino acid sequence of the laccase GL-Lac is shown in SEQ ID NO.1;

[0015] The mycotoxins include at least one of aflatoxin B1, zearalenone, vomitoxin, and T-2 toxin.

[0016] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention adopts a composite induction strategy, Ganoderma lucidum A novel laccase (named GL-Lac) was obtained from the culture medium. The laccase has the amino acid sequence shown in SEQ ID NO: 1 and has the following properties:

[0018] The degradation rates of AFB1, ZEN, DON and T-2 toxins were ≥80%, ≥90%, ≥78% and ≥75%, respectively; non-phenolic toxins such as AFB1 could be efficiently oxidized without the need for external mediators. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0020] Example 1

[0021] Preparation and extraction of laccase GL-Lac

[0022] (1) Activation of bacteria: Take Ganoderma lucidum ( Ganoderma lucidum ACCC 53264 was inoculated onto PDA plates containing 0.5 mM CuSO4 and cultured at 28°C for 5 days until the mycelium filled the plate.

[0023] (2) Seed liquid culture: Take 5 pieces of 5 mm diameter cakes (active mycelial zone at the edge) from the activated plate and inoculate them into a 250 mL conical flask (containing 100 mL of wheat bran extract culture medium). Culture at 28°C and 150 rpm for 3 days. 600 End the culture at 1.2-1.5).

[0024] Wheat bran extract medium:

[0025] Wheat bran extract 15 g / L (boil for 30 minutes and filter to obtain the supernatant)

[0026] Ammonium tartrate 3 g / L

[0027] KH2PO41 g / L

[0028] MgSO4·7H2O 0.5 g / L

[0029] CuSO4·5H2O 0.125 g / L (0.5 mM)

[0030] pH 5.5 (adjusted with 0.1 M HCl or NaOH);

[0031] Wheat bran extract provides a complex carbon source (hemicellulose, lignin fragments) to simulate the natural degradation environment;

[0032] Low nitrogen (ammonium tartrate) promotes the accumulation of secondary metabolites such as laccase.

[0033] (3) Induction of enzyme production: Transfer the seed solution into the induction medium (50 mL / 250 mL conical flask) at a 10% inoculum volume, culture at 28°C and 200 rpm with shaking, and culture until the mid-logarithmic growth phase. Add the inducer composition (to avoid initial growth inhibition) and continue culturing. During this period, take samples and determine the laccase activity using the ABTS method. Stop culturing if the laccase activity is ≥500 U / L after 7 days of culture.

[0034] Induction medium (pH 5.0, adjusted before sterilization):

[0035] Corn flour 20 g / L (gelatinized at 80°C for 30 min and then filtered);

[0036] Peptone 2 g / L;

[0037] KH2PO4 1 g / L;

[0038] MgSO4·7H2O 0.5 g / L;

[0039] The following inducer composition was added per 1 L of culture medium: 0.3 mM coumarin, 0.5 mM acetosyringone, 2 mM Cu 2+ , 0.05 mM β-estradiol, 2.0 g / L lignin sulfonate, 0.10 mM Mn 2+ , 10 μg / L 3-acetyl-DON, and 0.5 mM ferulic acid.

[0040] Laccase purification:

[0041] 1) Preparation of crude enzyme solution: Centrifuge the culture solution at 8000 rpm at 4°C for 15 min. Filter the supernatant through a 0.45 μm filter to obtain the crude enzyme solution.

[0042] 2) Ammonium sulfate fractional precipitation: Take the crude enzyme solution prepared in step 1) and slowly add solid (NH4)2SO4 to 30% saturation. Then, stir at 4°C for 1 hour. Centrifuge at 10,000 rpm for 20 minutes and discard the precipitate (impurities). Take the supernatant and add (NH4)2SO4 to 70% saturation. Collect the precipitate by centrifugation. Redissolve the precipitate in 10 mM sodium citrate buffer (pH 5.0) (containing 0.1 mM CuSO4) and dialyze for desalination.

[0043] 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 from 0 to 1 M NaCl was used to collect the laccase activity peak (0.3-0.5 M NaCl range); Concentration: Ultrafiltration centrifugation (10 kDa molecular weight cutoff) was used to obtain laccase GL-Lac.

[0044] ) Purity verification: SDS-PAGE: single band (65 kDa), purity ≥95% (gel scanning analysis); specific activity: ≥50 U / mg (ABTS method, pH 3.0).

[0045] The amino acid sequence of laccase GL-Lac was analyzed, and its amino acid sequence is as follows:

[0046] SRHWHGFFQEGSSWADGPVGVTQCPIAPGDSFLYRFKVPDQAGTFWYHSMAKFQSLLSCVTLLFAASAHAGIGPKADLTISNANIAPDGYTRAAVVVNGVFPGPLITGNKGDRFQLNVIDQLTNHTMLKTTSIHWHGFF QKGTNWADGPAFINQCPIASGHSFLYDFQVPDQAGTFWYHSHLSTQYCDGLRGPFVVYDPKDPLKGLYDVDNDSTVITLSDWYHVAARLGPSFPLGSDSTLINGLGRSTTNATAGLAVINVTQGKRYRFRLVSLSCDPNY TFSIDGHDSSVIEADGIATQPVTANAIQIFSAQRYSFVLTKNQTIGNYWIRANPSFGNIGFTTGINSAILRYSGADPIEPTTAQQTTQNLLNEVDLHPFVAMQTPGRATQGGTDVAINMVFNFNGSNFFINNASFTPPTV PVLLQILSGAQAAQDLLPSGSVYTLPINKSSELTFPATVNAPGAPHPFHLHHSFAVVRSAGSTEYNYNNPVWRDVVSTGTPAAGDNVTIRFQTDNPGPWFLHCHIDFHLEAGFAVVFAEDTADTSLANHVPQAWSDLCP TYDALSADDH, SEQ ID NO.1.

[0047] Example 2

[0048] Laccase GL-Lac degrades mycotoxins

[0049] GL-Lac (500 U / L) was reacted with toxins (1 μg / mL AFB1 / ZEN / DON / T-2) at pH 4.5 and 40°C for 24 h. The degradation rates of different toxins were measured. The specific experimental procedures are as follows:

[0050] Preparation of laccase stock solution: purified GL-Lac laccase (specific activity ≥50 U / mg, dissolved in 10 mM sodium citrate buffer, pH 5.0);

[0051] Preparation of toxin stock solution:

[0052] aflatoxin B1 (AFB1, 1 μg / mL dissolved in methanol);

[0053] zearalenone (ZEN, 1 μg / mL dissolved in acetonitrile);

[0054] vomitoxin (DON, 1 μg / mL dissolved in water);

[0055] T-2 toxin (1 μg / mL in methanol);

[0056] Reaction buffer: 0.1 M citrate-phosphate buffer (pH 4.5).

[0057] The detection system settings are shown in Table 1:

[0058] Table 1 Detection system settings

[0059]

[0060] Note: Dry the toxin stock solution with nitrogen and re-dissolve it in buffer (to avoid organic solvents inhibiting enzyme activity).

[0061] Control group: No laccase was added (buffer + toxin only). This was used to calculate the degradation rate, with three replicates per group. The above reaction system was added to a 1.5 mL centrifuge tube, vortexed to mix, and placed in a constant temperature water bath shaker at 40°C and 150 rpm for 24 h. The reaction was terminated by adding 100 μL of 1 M HCl (pH < 2.0), immediately placed in an ice bath, and then centrifuged at 4°C and 12,000 rpm for 5 min. The supernatant was used to detect the degradation rate of the target toxin. The specific detection method is as follows:

[0062] (1) AFB1 degradation rate (HPLC-FLD detection)

[0063] Chromatographic conditions:

[0064] Chromatographic column: C18 column (4.6×250 mm, 5 μm);

[0065] Mobile phase: water:methanol:acetonitrile (60:20:20, v / v / v);

[0066] Flow rate: 1 mL / min;

[0067] Detector: Fluorescence (Ex 360 nm, Em 440 nm);

[0068] Calculation: Degradation rate (%) = (1-sample peak area / control peak area) × 100.

[0069] (2) ZEN degradation rate (ELISA test)

[0070] Steps: Follow the instructions of the RIDASCREEN® ZEN kit to determine the residual ZEN concentration in the reaction solution;

[0071] Standard curve range: 0.1-5 μg / L;

[0072] Calculation: Degradation rate (%) = (1-sample OD value / control OD value) × 100.

[0073] (3) Degradation rate of DON / T-2 toxin (HPLC-UV detection)

[0074] Chromatographic conditions:

[0075] Detection wavelength: DON (220 nm), T-2 (210 nm);

[0076] Mobile phase: acetonitrile:water (15:85, v / v);

[0077] Calculation: Same as AFB1.

[0078] The test results are shown in Table 2.

[0079] Table 2 Degradation rate and residual rate of different toxins

[0080]

[0081] Result analysis: As shown in Table 2, the degradation rates of AFB1, ZEN, DON and T-2 toxins prepared by the laccase in the present application were ≥80%, ≥90%, ≥78% and ≥75%, respectively; the non-phenolic toxin AFB1 could be efficiently oxidized without the addition of an external mediator.

[0082] Control group 1

[0083] The preparation method of laccase was the same as that of Example 1, except that no inducing composition was added during the culture process.

[0084] The degradation effect and residual rate of the product of control group 1 on different toxins were further determined using the same method as in Example 2. The results are shown in Table 3.

[0085] Table 3 Degradation rate and residual rate of different toxins

[0086]

[0087] Analysis of results: It can be seen from Table 3 that even without adding the inducing composition, the degradation rates of the product for AFB1, ZEN, DON and T-2 toxins reached ≥70%, ≥80%, ≥60% and ≥60%, respectively; the non-phenolic toxin AFB1 can be efficiently oxidized without the addition of an external mediator.

[0088] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0089] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A biological preparation for degrading mycotoxins, characterized in that: The invention comprises laccase GL-Lac; the amino acid sequence of the laccase GL-Lac is shown as SEQ ID NO.

1.

2. The mycotoxin-degrading biological agent according to claim 1, characterized in that: The mycotoxin is 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.

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

4. The mycotoxin-degrading biological agent according to claim 3, characterized in that: The laccase GL-Lac is prepared by the following method: culturing Ganoderma lucidum ACCC 53264 in an induction medium to mid-logarithmic growth phase, adding an inducer composition, performing induction culture, and centrifuging, separating, and precipitating to prepare the laccase GL-Lac.

5. The mycotoxin-degrading biological preparation according to claim 4, characterized in that: The composition and ratio of the induction medium are as follows: 20 g / L corn flour, 2 g / L peptone, 1 g / L KH2PO4 and 0.5 g / L MgSO4·7H2O; pH 5.

0.

6. The mycotoxin-degrading biological agent according to claim 5, characterized in that: The following inducer composition was added per 1 L of culture medium: 0.3 mM coumarin, 0.5 mM acetosyringone, 2 mM Cu 2+ , 0.05 mM β-estradiol, 2.0 g / L lignin sulfonate, 0.10 mM Mn 2+ , 10 μg / L 3-acetyl-DON, and 0.5 mM ferulic acid.

7. The mycotoxin-degrading biological agent according to claim 1, characterized in that: Other active ingredients and / or other physiologically acceptable carriers may also be included.

8. Use of the biological preparation according to any one of claims 1 to 7 for degrading mycotoxins in feed; the mycotoxin is at least one of aflatoxin B1, zearalenone, vomitoxin, and T-2 toxin.

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

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