Application of corn-based ethanol fermentation byproduct extract as an efficient mediator for laccase oxidation of aflatoxin B1

Abstract

This invention discloses the use of a corn-based ethanol fermentation byproduct extract as a highly efficient mediator for the laccase oxidative conversion of aflatoxin B1. Laccase oxidizes and converts aflatoxin B1 in the presence of the corn-based ethanol fermentation byproduct extract. This invention not only overcomes the potential secondary contamination of traditional mediators with potential toxicity but also significantly improves the efficiency of laccase oxidative conversion of aflatoxin B1, potentially enabling widespread use in the field of mycotoxin detoxification in food and feed.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to an application of a corn-based ethanol fermentation byproduct extract as a high-efficiency mediator for laccase oxidation and conversion of aflatoxin B1. Background Art

[0002] Aflatoxins are a class of compounds with very similar chemical structures produced by molds such as Aspergillus flavus and Aspergillus parasiticus. They are the most important mycotoxins that contaminate grain and oil crops, food, and feed in my country and worldwide. They are highly toxic and carcinogenic. The most important of these is aflatoxin B1, which has been classified as a Class I carcinogen by the World Health Organization and poses a serious threat to human and animal health. Therefore, it is urgent to develop a simple, effective, and environmentally friendly method for detoxifying aflatoxin B1.

[0003] The detoxification methods for aflatoxin B1 reported in current research include physical, chemical, and biological methods. Physical and chemical detoxification methods have disadvantages such as operational difficulties, unstable results, significant nutrient loss, impact on feed palatability, and the potential for secondary contamination. Biological methods are mainly divided into two categories: one uses microbial cells and cell walls to adsorb the toxin, and the other uses enzyme proteins identified and isolated from microorganisms, animals, and plants to degrade the toxin. The latter utilizes enzyme catalysis, which is characterized by high efficiency, high stereoselectivity, and mild reaction conditions, making it an important and effective method for detoxifying aflatoxin B1.

[0004] Laccase is a copper-containing polyphenol oxidase that catalyzes the oxidation of various phenolic and non-phenolic substances, simultaneously reducing oxygen to water with electron transfer. It is therefore known as a "green catalyst." Due to its wide availability and low cost, laccase is widely used in mycotoxin detoxification research. However, existing laccases are generally inefficient in directly catalyzing the degradation of mycotoxins due to their low redox potential. By adding a redox mediator to the reaction system of laccase and mycotoxins, the mediator is oxidized by laccase, further oxidizing the mycotoxins with high redox potentials. Currently, commonly used mediators can be divided into three categories: 1) synthetic mediators, such as 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), violaceous acid (VIO), 1-hydroxybenzotriazole (HBT), and 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO); 2) natural mediators, such as syringaldehyde (SA), acetosyringone (AS), acetovanillone, and p-coumaric acid; and 3) other mediators, such as polyoxometalates (POM). However, synthetic mediators have the following disadvantages: 1) poor stability; 2) high cost; 3) potential toxicity; some mediators, upon oxidation, produce toxic byproducts that inactivate enzymes; and 4) most synthetic mediators cannot be regenerated after oxidation. These shortcomings limit their application. Natural mediators are not only easily available and inexpensive but also environmentally friendly and have greater economic value. Therefore, extracting low-cost electron transfer mediators from natural materials will be an important direction for the development of laccase biodetoxification technology. Summary of the Invention

[0005] This invention provides the use of a corn-based ethanol fermentation byproduct extract as a highly efficient mediator for the laccase oxidation and conversion of aflatoxin B1. This method utilizes a corn-based ethanol fermentation byproduct extract as a redox mediator, effectively reducing the amount of laccase required while improving the oxidation and conversion efficiency of aflatoxin B1. This method overcomes the secondary contamination problem caused by traditional mediators and has promising application prospects in the field of mycotoxin detoxification in food and feed.

[0006] The present invention uses the corn-based ethanol fermentation byproduct extract as a high-efficiency mediator for laccase to oxidize and convert aflatoxin B1. The corn-based ethanol fermentation byproduct extract is used as a mediator, and laccase oxidizes and converts aflatoxin B1 in the presence of the mediator.

[0007] The process of laccase oxidative conversion of aflatoxin B1 is carried out in a citric acid-disodium hydrogen phosphate solution with a concentration of 50 mM and a pH of 4.0-8.0.

[0008] During the laccase oxidation and conversion of aflatoxin B1, the amount of laccase added is 2.5-50 mU / mL; the amount of corn-based ethanol fermentation byproduct extract added is 0.4-10 mg / L.

[0009] The laccase is a white rot fungus laccase, and the enzyme code is EC 1.10.3.2.

[0010] The corn-based ethanol fermentation byproduct extract is prepared by a method comprising the following steps:

[0011] (1) Referring to the conventional dry-grinding corn-based ethanol processing process, the corn-based ethanol fermentation broth is distilled to obtain ethanol product and distillation by-products, and the by-products are centrifuged to separate solid matter and thin distillate;

[0012] (2) extracting the thin distillate from step (1) by mixing equal volumes of ethyl acetate as an extractant three times, and combining the extracts;

[0013] (3) subjecting the extract obtained in step (2) to rotary evaporation;

[0014] (4) dissolving the product after rotary evaporation in step (3) with methanol, and blowing dry with a nitrogen blower to obtain a corn-based ethanol fermentation byproduct extract.

[0015] In step (1), the centrifugation is carried out at room temperature and 1000-2000 rpm for 5-10 minutes.

[0016] In step (2), the thin distillate is mixed with an extractant, ethyl acetate, in equal volumes and extracted three times, and the extracts are combined.

[0017] In step (3), rotary evaporation is performed at a temperature of 20-40° C. and a rotation speed of 50-60 rpm.

[0018] The mediator of the present invention can efficiently oxidize and convert aflatoxin B1, is low-cost, and has a wide range of applications, and can be widely used in the field of feed toxin-degrading enzymes. The mycotoxin detoxification method provided by the present invention is simple to operate, highly efficient, low-cost, and highly safe, and has important practical and promotional value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The oxidative transformation of aflatoxin B1 by different laccase addition amounts in the laccase-corn-based ethanol fermentation byproduct extract system was shown;

[0020] Figure 2 The oxidative transformation of aflatoxin B1 by different mediator addition amounts in the laccase-corn-based ethanol fermentation byproduct extract system was shown;

[0021] Figure 3HPLC analysis results showing the oxidative conversion of aflatoxin B1 by the laccase-corn-based ethanol fermentation byproduct extract system. DETAILED DESCRIPTION

[0022] The implementation methods in the following examples are all conventional methods unless otherwise specified.

[0023] Example 1:

[0024] Referring to the conventional dry-grinding corn-based ethanol processing process, corn was added to a grinder for crushing, 96g of corn flour was taken and added to a 500mL triangular conical flask, 204g of water was added thereto, and after stirring evenly, the pH was adjusted to 5.7-5.8 with sulfuric acid, and 0.02g of α-amylase was added thereto. The mixture was kept warm in a water bath shaker at 90°C and 180rpm for 2h; then the temperature was lowered to about 35°C, the pH was adjusted to about 4.2-4.3 with sulfuric acid, and then 0.08g of glucose amylase was added for saccharification for 4h. Finally, 0.05g of urea and 0.3g of dry yeast were added to the saccharified mash, and the mixture was placed in a shaker at 32°C and 120rpm for fermentation for 60h. The corn-based ethanol fermentation broth is distilled to separate the ethanol product and distillation byproducts. 200 g of the distillation byproduct is centrifuged at room temperature at 1000-2000 rpm for 5-10 minutes to obtain the supernatant, the thin distillate. The thin distillate is extracted three times with equal volumes of ethyl acetate, and the extracts are combined. The extracts are transferred to a round-bottom flask and rotary evaporated at 20-40°C and 50-60 rpm. Approximately 10 mL of methanol is added to the flask, mixed thoroughly, and poured into a 50 mL centrifuge tube. The mixture is then blown dry with a nitrogen blower to obtain the corn-based ethanol fermentation byproduct extract.

[0025] Example 2: Oxidative conversion of aflatoxin B1 by laccase at different concentrations using corn-based ethanol fermentation byproduct extract as a medium

[0026] Aflatoxin B1 was dissolved in dimethyl sulfoxide to a 100 mg / L stock solution. The following reaction system was used: 960 μL of 50 mM citric acid-disodium hydrogen phosphate solution, pH 7.0, 10 μL of the aflatoxin B1 stock solution, 20 μL of a corn-based ethanol fermentation byproduct extract (0.5 mg / mL), and 10 μL of laccase at varying concentrations (0.25-5.0 U / mL). A control without laccase was used, and the reactions were repeated three times. The reaction was carried out at 45°C and terminated after 12 hours by adding an equal volume of methanol. The oxidation conversion efficiency of aflatoxin B1 was analyzed by high-performance liquid chromatography. The liquid chromatography was performed on an Agilent 1260 high performance liquid chromatography analysis system, the chromatographic separation column was a ZORBAX SB-C18 (4.6 mm × 150 mm, 5 μm), the mobile phase was methanol-water (45-55), the flow rate was 0.8 ml / min, and the detection wavelengths of the fluorescence detector were λex = 360 nm, λem = 440 nm.

[0027] The results are as follows Figure 1 As shown in the results, the oxidation conversion efficiency of aflatoxin B1 gradually increased with the increase of laccase addition, and then tended to be stable. When the addition amount of laccase increased from 2.5mU / mL to 50mU / mL, the oxidation conversion efficiency of aflatoxin B1 increased from 41.3% to 83.5%.

[0028] Example 3: Laccase Oxidative Conversion of Aflatoxin B1 Using Different Concentrations of Corn-Based Ethanol Fermentation Byproduct Extracts

[0029] Aflatoxin B1 was dissolved in dimethyl sulfoxide to a 100 mg / L stock solution. The following reaction system was used: 960 μL of 50 mM citric acid-disodium hydrogen phosphate solution, pH 7.0, 10 μL of the aflatoxin B1 stock solution, 20 μL of corn-based ethanol fermentation byproduct extract at varying concentrations (0-0.5 mg / mL), and 10 μL of laccase (2.5 U / mL). A control without laccase was used, and the reaction was repeated three times. The reaction was carried out at 45°C and terminated after 12 hours by adding an equal volume of methanol. The oxidation conversion efficiency of aflatoxin B1 was analyzed by high-performance liquid chromatography.

[0030] The results are as follows Figure 2 and Figure 3As shown, the oxidation efficiency of aflatoxin B1 gradually increased with increasing amounts of corn-based ethanol fermentation byproduct extract. In the absence of a mediator, laccase achieved only a 6.5% oxidation efficiency for aflatoxin B1. However, when the mediator addition level was increased to 10 mg / L, the efficiency increased to 84.3%. This represents an approximately 13-fold increase in oxidation efficiency compared to the absence of a mediator. Therefore, the addition of corn-based ethanol fermentation byproduct extract as a mediator significantly enhanced the oxidation efficiency of laccase for aflatoxin B1.

Claims

1. The use of a corn-based ethanol fermentation byproduct extract as a highly efficient mediator for the laccase oxidation conversion of aflatoxin B1, characterized by: A corn-based ethanol fermentation byproduct extract is used as a mediator, and laccase oxidizes and converts aflatoxin B1 in the presence of the mediator; The laccase oxidation and conversion of aflatoxin B1 was carried out in a citric acid-disodium hydrogen phosphate solution with a concentration of 50 mM and a pH of 4.0-8.0; During the laccase oxidation and conversion of aflatoxin B1, the amount of laccase added was 2.5-50 mU / mL; the amount of corn-based ethanol fermentation byproduct extract added was 0.4-10 mg / L; The corn-based ethanol fermentation byproduct extract is prepared by a method comprising the following steps: (1) The corn-based ethanol fermentation broth is distilled to obtain ethanol product and distillation by-products, and the by-products are centrifuged to separate solid matter and thin distillate; (2) extracting the thin distillate from step (1) by mixing equal volumes of ethyl acetate as an extractant three times, and combining the extracts; (3) subjecting the extract obtained in step (2) to rotary evaporation; (4) Dissolve the product after rotary evaporation in step (3) in methanol and blow dry with a nitrogen blower to obtain a corn-based ethanol fermentation byproduct extract.

2. The use according to claim 1, characterized in that: In step (3), rotary evaporation is performed at a temperature of 20-40° C. and a rotation speed of 50-60 rpm.

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